Soluble TNF Receptors and Their Use in Treatment of Disease

ABSTRACT

The present invention relates to tumor necrosis factor (TNF) antagonists and corresponding nucleic acids derived from tumor necrosis factor receptors (TNFRs) and their use in the treatment of inflammatory diseases. These proteins are soluble secreted decoy receptors that bind to TNF and prevent TNF from signaling to cells. In particular, the proteins are mammalian TNFRs that lack exon 7 and which can bind TNF and can act as a TNF antagonist.

This application is a continuation-in-part of U.S. application Ser. No.11/595,485, filed Nov. 10, 2006 which claims priority to U.S.Provisional application Ser. No. 60/862,350, filed Oct. 20, 2006, andU.S. Provisional application Ser. No. 60/735,429, filed Nov. 10, 2005,all of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to tumor necrosis factor (TNF) antagonistsand corresponding nucleic acids derived from TNF receptors and their usein the treatment of inflammatory diseases. These proteins are solublesecreted decoy receptors that bind to TNF-α and prevent TNF-α fromsignaling to cells.

BACKGROUND OF THE INVENTION

TNF-α is a pro-inflammatory cytokine that exists as a membrane-boundhomotrimer and is released as a homotrimer into the circulation by theprotease TNF-α converting enzyme (TACE). TNF-α is introduced into thecirculation as a mediator of the inflammatory response to injury andinfection. TNF-α activity is implicated in the progression ofinflammatory diseases such as rheumatoid arthritis, Crohn's disease,ulcerative colitis, psoriasis and psoriatic arthritis (Palladino, M. A.,et al., 2003, Nat. Rev. Drug Discov. 2:736-46). Acute exposure to highTNF-α levels, as experienced during a massive infection, results insepsis. Its symptoms include shock, hypoxia, multiple organ failure, anddeath. Chronic low-level release of TNF-α is associated withmalignancies and leads to cachexia, a disease characterized by weightloss, dehydration and fat loss.

TNF-α activity is mediated primarily through two receptors coded by twodifferent genes, TNF-α receptor type I (hereafter “TNFR1”, exemplifiedby GenBank accession number X55313 for human TNFR1) and TNF-α receptortype II (hereafter “TNFR2”, exemplified by GenBank accession number NM001066 for human TNFR2). TNFR1 is a membrane-bound protein with amolecular weight of approximately 55 kilodaltons (kDal), while TNFR2 isa membrane-bound protein with a molecular weight of approximately 75kDal. TNFR1 and TNFR2 belong to a family of receptors known as the TNFreceptor (TNFR) superfamily. The TNFR superfamily is a group of type Itransmembrane proteins, with a carboxy-terminal intracellular domain andan amino-terminal extracellular domain characterized by a commoncysteine rich domain (CRD). TNFR1 and TNFR2 have a unique domain incommon, called the pre-ligand-binding assembly domain (PLAD) that isrequired for assembly of multiple receptor subunits and subsequentbinding to TNF-α.

TNFR1 and TNFR2 also share a common gene structure, in which the codingsequence of each extends over 10 exons separated by 9 introns (Fuchs, etal., 1992, Genomics 13:219; Santee, et al., 1996, J. Biol. Chem.35:21151). Most of the transmembrane domain sequence is encoded by theseventh exon (“exon 7”) (See FIG. 1).

Experiments in knockout mice lacking both TNFR1 and TNFR2 demonstratedthat the injury-induced immune response to brain injury was suppressed,suggesting that drugs that target the TNF signaling pathways may bebeneficial in treating stroke or traumatic brain injury (Bruce, et al.,1996, Nat. Med. 2:788). TNFR2 knockout mice, but not TNFR1 knockoutmice, were resistant-to experimentally-induced cerebral malaria (Lucas,R., et al., 199-7, Eur. J. Immunol. 27:1719); whereas TNFR1 knockoutmice were resistant to autoimmune encephalomyelitis (Suvannavejh, G. C.,et al., 2000, Cell. Immunol., 205:24). These knockout mice are modelsfor human cerebral malaria and multiple sclerosis, respectively.

TNFR2 is present at high density on T cells of patients withinterstitial lung disease, suggesting a role for TNFR2 in the immuneresponses that lead to alveolitis (Agostini, C., et al., 1996, Am. J.Respir. Crit. Care Med, 153:1359). TNFR2 is also implicated in humandisorders of lipid metabolism. TNFR2 polymorphism is associated withobesity and insulin resistance (Fernandez-Real, et al., 2000, DiabetesCare, 23:831), familial combined hyperlipidemia (Geurts, et al., 2000,Hum. Mol. Genet 9:2067), hypertension and hypercholesterolemia (Glenn,et al., 2000, Hum. Mol. Genet., 9:1943). In addition, TNFR2 polymorphismis associated with susceptibility to human narcolepsy (Hohjoh, H., etal., 2000, Tissue Antigens, 56:446) and to systemic lupus erythematosus(Komata, T., et al., 1999, Tissue Antigens, 53:527).

To simplify further analysis and comparison, the human TNFR2 461 aminoacid sequence provided in SEQ ID No: 4, GenBank accession numberNP_001057, is used as a reference unless stated otherwise (FIG. 1).Amino acid 1 is the first amino acid of the full length protein humanTNFR2, which includes the signal sequence. Amino acid 23 located in exon1 is the first amino acid of the mature protein, which is the proteinafter cleavage of the signal sequence. The transmembrane region spansamino acids 258-287. The exon 6/7 junction is located within the codonthat encodes residue 263, while the exon 7/8 junction is located withinthe codon that encodes residue 289.

Physiological, soluble fragments of both TNFR1 and TNFR2 have beenidentified. For example, soluble extracellular domains of thesereceptors are shed to some extent from the cell membrane by the actionof metalloproteases (Palladino, M. A., et al., 2003, Nat. Rev. DrugDiscov. 2:736-46). Additionally, the pre-mRNA of TNFR2 undergoesalternative splicing, creating either a full length, activemembrane-bound receptor, or a secreted receptor that lacks exons 7 and 8(Lainez et al., 2004, Int. Immunol., 16:169) (“Lainez”). The secretedprotein binds TNF-α but does not elicit a physiological response, hencereducing overall TNF-α activity. Although an endogenous, secreted splicevariant of TNFR1 has not yet been identified, the similar genomicstructure of the two receptors suggests that a TNFR1 splice variant canbe produced.

The cDNA for the splice variant identified by Lainez contains the 113 bpdeletion of exons 7 and 8. This deletion gives rise to a stop codon 17bp after the end of exon 6. Consequently, the protein has the sequenceencoded by the first six exons of the TNFR2 gene (residues 1-262)followed by a 6 amino acid tail of Ala-Ser-Leu-Ala-Cys-Arg.

Additional soluble fragments of recombinantly-engineered TNF receptorsare known. In particular, truncated forms of TNFR1 or TNFR2 have beenproduced which have (1) all or part of the extracellular domain or (2) aTNFR extracellular domain fused to another protein.

Smith discloses truncated human TNFR2s, including a protein withresidues 23-257, which terminates immediately before the transmembraneregion, and a protein with residues 23-185 (U.S. Pat. No. 5,945,397).Both TNFR2 fragments are soluble and capable of binding TNF-α.

Craig discloses that an extracellular domain of human TNFR2 withresidues 23-257 fused to the Fc region of human IgG₁ (TNFR:Fc) is aTNF-α antagonist capable of reducing inflammation in rat and micearthritis models (U.S. Pat. No. 5,605,690). TNFR:Fc is an FDA-approvedtreatment for certain forms of arthritis, ankylosing spondylitis, andpsoriasis and is sold under the name etanercept (Enbrel®).

Moosmayer demonstrated that soluble human TNFR2 proteins containing theentire intracellular domain are more active TNF antagonists than theextracellular domain alone (Moosmayer et al., 1996, J. InterferonCytokine Res., 16:471). In those experiments, Moosmayer compared theactivities of solubilized full length TNFR2 (1-461), with TNFR2 lackingall but the three C-terminal amino acids of the transmembrane region(ATM) (1-258 joined to 283-461), TNFR extracellular domain (1-258), andTNFR:Fc. The inhibition of TNF-mediated cytotoxicity by the ATM proteinand solubilized full length TNFR2 are comparable. However, theiractivities are approximately 60-fold higher than the TNFR2 extracellulardomain alone, but approximately seven-fold less than TNFR:Fc.

Since excess TNF-α activity is associated with disease pathogenesis,particularly for inflammatory conditions, there is a need for TNF-αantagonists and methods for their use in the treatment of inflammatorydiseases. Concerns have been raised regarding the side effects ofcurrently approved protein-based TNF-α antagonists, including TNFR:Fc;these concerns include exacerbation of latent tuberculosis, worsening ofcongestive heart failure, and increased risk of lymphoma (Palladino, M.A., et al., 2003, Nat. Rev. Drug Discov. 2:736-46). Furthermore, thereare patients who do not respond to currently approved TNF-α antagonists.Therefore, there is a continuing need to identify new TNF-α antagonists.

To that end, Sazani et al. have shown, inter alia, that by using spliceswitching oligonucleotides (SSOs) it is possible to generatealternatively spliced mRNA coding for variant TNFR1 or TNFR2 proteinsusing the naturally-occurring exon and intron structure (U.S.application Ser. No. 11/595,485). In particular, the SSOs lead the cellto produce mRNAs that encode novel TNFR proteins that lack only exon 7,which encodes most of the transmembrane region of these proteins.Further characterization of the TNFR2 protein lacking only exon 7surprisingly showed that it is a particularly stable, soluble decoyreceptor that binds to and inactivates extracellular TNF-α. This proteinunexpectedly has anti-TNF-α activity that is at least equivalent toTNFR:Fc.

SUMMARY OF THE INVENTION

One embodiment of the invention is a protein, either full length ormature, which can bind TNF, is encoded by a cDNA derived from amammalian TNFR gene, and in the cDNA exon 6 is followed directly by exon8 and as a result lacks exon 7 (“TNFR Δ7”). In another embodiment, theinvention is a pharmaceutical composition comprising a TNFR Δ7. In afurther embodiment, the invention is a method of treating aninflammatory disease or condition by administering a pharmaceuticalcomposition comprising a TNFR Δ7.

In yet another embodiment, the invention is a nucleic acid that encodesa TNFR Δ7. In a further embodiment, the invention is a pharmaceuticalcomposition comprising a nucleic acid that encodes a TNFR Δ7.

In another embodiment, the invention is an expression vector comprisinga nucleic acid that encodes a TNFR Δ7. In a further embodiment, theinvention is a method of increasing the level of a soluble TNFR in theserum of a mammal by transforming cells of the mammal with an expressionvector comprising a nucleic acid that encodes a TNFR Δ7.

In another embodiment, the invention is a cell transformed with anexpression vector comprising a nucleic acid that encodes a TNFR Δ7. In afurther embodiment, the invention is a method of producing a TNFR Δ7 byculturing, under conditions suitable to express the TNFR Δ7, a celltransformed with an expression vector comprising a nucleic acid thatencodes a TNFR Δ7. In yet another embodiment, the invention is a methodof treating an inflammatory disease or condition by administering anexpression vector comprising a nucleic acid that encodes a TNFR Δ7.

In yet another embodiment, splice-switching oligomers (SSOs) aredisclosed that alter the splicing of a mammalian TNFR2 pre-mRNA toproduce a mammalian TNFR2 protein, which can bind TNF and where exon 6is followed directly by exon 8 and as a result lacks exon 7 (“TNFR2Δ7”). One embodiment of the invention is a method of treating aninflammatory disease or condition by administering SSOs to a patient ora live subject. The SSOs that are administered alter the splicing of amammalian TNFR2 pre-mRNA to produce a TNFR2 Δ7. In another embodiment,the invention is a method of producing a TNFR2 Δ7 in a cell byadministering SSOs to the cell.

The foregoing and other objects and aspects of the present invention arediscussed in detail in the drawings herein and the specification setforth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts the human TNFR2 structure. Relevant exonsand introns are represented by boxes and lines, respectively. The signalsequence and the transmembrane region are shaded. Residues that form theboundaries of the signal sequence, the transmembrane region, and thefinal residue are indicated below the diagram. Exon boundaries areindicated above the diagram; if the 3′ end of an exon and the 5′ end ofthe following exon have the same residue number, then the splicejunction is located within the codon encoding that residue.

FIG. 2A graphically illustrates the amount of soluble TNFR2 from SSOtreated primary human hepatocytes. The indicated SSO was transfectedinto primary human hepatocytes at 50 nM. After ˜48 hrs, theextracellular media was analyzed by enzyme linked immunosorbant assay(ELISA) for soluble TNFR2 using the Quantikine® Human sTNF RII ELISA kitfrom R&D Systems (Minneapolis, Minn.). Error bars represent the standarddeviation for 3 independent experiments.

FIG. 2B: Total RNA was analyzed for TNFR2 splice switching by RT-PCRusing primers specific for human TNFR2. SSOs targeted to exon seven ledto shifting from full length TNFR2 mRNA (FL) to TNFR2 Δ7 mRNA (Δ7). SSO3083 is a control SSO with no TNFR2 splice switching ability.

FIG. 3 shows the splicing products of L929 cells treated with SSO10-mers targeted to mouse TNFR2 exon 7. L929 cells were transfected withthe indicated SSO concentration (50 or 100 nM), and evaluated for spliceswitching of TNFR2 by RT-PCR 24 his later. PCR primers were used toamplify from Exon 5 to Exon 9, so that “Full Length” (FL) TNFR2 isrepresented by a 486 bp band. Transcripts lacking exon 7 (Δ7) isrepresented by a 408 bp band.

FIGS. 4A and 4B show the splicing products of mice treated with SSO10-mers targeted to mouse TNFR2 exon 7. The indicated SSOs wereresuspended in saline, and injected i.p. into mice at 25 mg/kg/day for 5days. Mice were prebled before SSO injection, and 10 days after thefinal SSO injection and sacrificed. At the time of sacrifice, total RNAfrom livers was analyzed for TNFR2 splice switching by RT-PCR. FL—fulllength TNFR2; Δ7-TNFR2 Δ7 (FIG. 4A). The concentration of TNFR2 Δ7 inthe serum taken before (Pre) and after (Post) SSO injection wasdetermined by ELISA using the Quantikine® Mouse sTNF RII ELISA kit fromR&D Systems (Minneapolis, Minn.) (FIG. 4B). Error bars represent thestandard error from 3 independent readings of the same sample.

FIG. 5 depicts the splice switching ability of SSOs of differentlengths. Primary human hepatocytes were transfected with the indicatedSSO and TNFR2 expression analyzed by RT-PCR (top panel) and ELISA(bottom panel) as in FIG. 2. Error bars represent the standard deviationfrom 2 independent experiments.

FIGS. 6A and 6B illustrate TNFR2 Δ7 mRNA induction in the livers of SSOtreated mice. FIG. 6A: Total RNA from the livers of SSO 3274 treatedmice were subjected to RT-PCR, and the products visualized on a 1.5%agarose gel. The sequence of the exon 6-exon 8 junction is shown in FIG.6B.

FIGS. 7A and 7B illustrate TNFR2 Δ7 mRNA induction in SSO treatedprimary human hepatocytes. FIG. 7A: Total RNA from SSO 3379 treatedcells were subjected to RT-PCR, and the products visualized on a 1.5%agarose gel. The sequence of the exon 6-exon 8 junction is shown in FIG.7B.

FIGS. 8A and 8B illustrate the dose dependence of TNFR2 pre-mRNAsplicing shifting by SSO 3378, 3379 and 3384. Primary human hepatocyteswere transfected with 1-150 nM of the indicated SSO. After ˜48 hrs, thecells were harvested for total RNA, and the extracellular media wascollected. FIG. 8A: Total RNA was analyzed for TNFR2 splice switching byRT-PCR using primers specific for human TNFR2. For each SSO, amount ofsplice switching is plotted as a function of SSO concentration. FIG. 8B:The concentration of soluble TNFR2 in the extracellular media wasdetermined by ELISA and plotted as a function of SSO. Error barsrepresent the standard deviation for at least 2 independent experiments.

FIG. 9 graphically illustrates detection of secreted TNFR2 splicevariants from L929 cells. Cells were transfected with the indicatedSSOs, After 72 hrs, the extracellular media was removed and analyzed byELISA. The data are expressed as pg soluble TNFR2 per mL.

FIG. 10 shows the splicing products for intraperitoneal (i.p.) injectionof SSO 3274 (top) and 3305 (bottom) in mice. SSO 3274 was injected i.p.at 25 mg/kg/day for either 4 days (4/1 and 4/10) or 10 days (10/1). Micewere sacrificed either 1 day (4/1 and 10/1) or 10 days (4/10) after thelast injection and total RNA from liver was analyzed by RT-PCR for TNFR2splice switching as described in FIG. 3. SSO 3305 was injected at theindicated dose per day for 4 days. Mice were sacrificed the next day andthe livers analyzed as with 3274 treated animals.

FIG. 11A graphically illustrates the amount of soluble TNFR2 in mouseserum 10 days after SSO treatment. Mice were injected i.p. with theindicated SSO or saline (n=5 per group) at 25 mg/kg/day for 10 days.Serum was collected 4 days before injections began and on the indicateddays after the last injection. Sera was analyzed by ELISA as describedin FIG. 2. At day 10, mice were sacrificed and livers were analyzed forTNFR2 splice switching by RT-PCR (FIG. 11B) as described in FIG. 10.

FIG. 12A graphically illustrates the amount of soluble TNFR2 in mouseserum 27 days after SSO treatment. Mice were treated as described inFIG. 11, except that serum samples were collected until day 27 after thelast injection. SSOs 3083 and 3272 are control SSOs with no TNFR2 spliceswitching ability. At day 27, mice were sacrificed and livers wereanalyzed for TNFR2 splice switching by RT-PCR (FIG. 12B) as described inFIG. 11.

FIGS. 13A and 13B graphically depict the anti-TNF-α activity in acell-based assay using serum from 550 treated mice, where serum sampleswere collected 5 days (FIG. 6A) and 27 days (FIG. 6B) after 550treatment. L929 cells were treated with either 0.1 ng/mL TNF-α, or TNF-αplus 10% serum from mice treated with the indicated SSO. Cell viabilitywas measured 24 hrs later and normalized to untreated cells.

FIG. 14 graphically compares the anti-TNF-α activity of serum from theindicated SSO oligonucleotide-treated mice to recombinant soluble TNFR2(rsTNFR2) extracellular domain from Sigma® and to Enbrel® using the cellsurvival assay described in FIG. 13.

FIGS. 15A and 15B compare the stability of muTNFR2 Δ7 protein (FIG. 15A)and mRNA (FIG. 15B). Mice were injected at 25 mg/kg/day daily witheither SSO 3272, SSO 3274 or SSO 3305 (n=5). Mice were bled on theindicated day after the last injection and the serum TNFR2 concentrationwas measured. Total RNA from mice sacrificed on the indicated day afterthe last injection of SSO was subjected to RT-PCR as described in FIG.10.

FIG. 16 plots TNFR2 Δ7 protein (dashed line) and mRNA (solid line)levels over time, as a percentage of the amount of protein or mRNA,respectively, 10 days after the last injection.

FIG. 17 graphically illustrates the dose dependant anti-TNF-α activityof TNFR2 Δ7 expressed in HeLa cells after transfection with TNFR2 Δ7mammalian expression plasmids. HeLa cells were transfected with theindicated mouse or human TNFR2 Δ7 plasmid and extracellular media wascollected after 48 hrs. The TNFR2 Δ7 concentration in the media wasdetermined by ELISA and serial dilutions were prepared. These dilutionswere assayed for anti-TNF-α activity by the L929 cytoxicity assay as inFIG. 14.

FIG. 18 shows expressed mouse (A) and human (B) TNFR2 Δ7 proteinisolated by polyacrylamide gel electrophoresis (PAGE). HeLa cells weretransfected with the indicated plasmid. After ˜48 hrs, the extracellularmedia was collected and concentrated, and cells were collected in RIPAlysis buffer. The proteins in the samples were separated by PAGE and awestern blot was performed using a C-terminal TNFR2 primary antibody(Abeam) that recognizes both the human and mouse TNFR2 Δ7 proteins.Media, extracellular media samples from HeLa cells transfected with theindicated plasmid; Lysate, cell lysate from Hela cells transfected withthe indicated plasmid. CM, control media from untransfected HeLa cells;CL, control cell lysates from untransfected HeLa cells. +, molecularweight markers (kDal).

FIG. 19 shows purified His-tagged human and mouse TNFR2 Δ7.Unconcentrated extracellular media containing the indicated TNFR2 Δ7protein was prepared as in FIG. 18. Approximately 32 mL of the media wasapplied to a 1 mL HisPur cobalt spin column (Pierce), and bound proteinswere eluted in 1 mL buffer containing 150 mM imidazole. Samples of eachwere analyzed by PAGE and western blot was performed as in FIG. 18. Themultiple bands in lanes 1144-4 and 1319-1 represent variablyglycosylated forms of TNFR2 Δ7.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “tumor necrosis factor receptor”, “TNFreceptor”, and “TNFR” refer to proteins having amino acid sequences ofor which are substantially similar to native mammalian TNF receptorsequences, and which are capable of binding TNF molecules. In thiscontext, a “native” receptor or gene for such a receptor, means areceptor or gene that occurs in nature, as well as thenaturally-occurring allelic variations of such receptors and genes.

The term “mature” as used in connection with a TNFR means a proteinexpressed in a form lacking a leader or signal sequence as may bepresent in full-length transcripts of a native gene.

The nomenclature for TNFR proteins as used herein follows the conventionof naming the protein (e.g., TNFR2) preceded by a species designation,e.g., hu (for human) or mu (for murine), followed by a Δ (to designate adeletion) and the number of the exon(s) deleted. For example, huTNFR2 Δ7refers to human TNFR2 lacking exon 7. In the absence of any speciesdesignation, TNFR refers generically to mammalian TNFR.

The term “secreted” means that the protein is soluble, i.e., that it isnot bound to the cell membrane. In this context, a form will be solubleif using conventional assays known to one of skill in the art most ofthis form can be detected in fractions that are not associated with themembrane, e.g., in cellular supernatants or serum.

The term “stable” means that the secreted TNFR form is detectable usingconventional assays by one of skill in the art, such as, western blots,ELISA assays in harvested cells, cellular supernatants, or serum.

As used herein, the terms “tumor necrosis factor” and “TNF” refer to thenaturally-occurring protein ligands that bind to TNF receptors. TNFincludes, but is not limited to, TNF-α and TNF-β.

As used herein, the term “an inflammatory disease or condition” refersto a disease, disorder, or other medical condition that at least in partresults from or is aggravated by the binding of TNF to its receptor.Such diseases or conditions include, but are not limited to, thoseassociated with increased levels of TNF, increased levels of TNFreceptor; or increased sensitization or deregulation of thecorresponding signaling pathway. The term also encompasses diseases andconditions for which known TNF antagonists have been shown useful.Examples of inflammatory diseases or conditions include, but are notlimited to, rheumatoid arthritis, juvenile rheumatoid arthritis,psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatorybowel disease (including Crohn's disease and ulcerative colitis),hepatitis, sepsis, alcoholic liver disease, and non-alcoholic steatosis.

As used herein, the term “hepatitis” refers to a gastroenterologicaldisease, condition, or disorder that is characterized, at least in part,by inflammation of the liver. Examples of hepatitis include, but are notlimited to, hepatitis associated with hepatitis A virus, hepatitis Bvirus, hepatitis C virus, or liver inflammation associated withischemia/reperfusion.

As used herein, the term “TNF antagonist” means that the protein iscapable of measurable inhibition of TNF-mediated cytotoxicity usingstandard assays as are well known in the art. (See, e.g., Example 1below, L929 cytotoxicity assay).

The term “binds TNF” means that the protein can bind detectable levelsof TNF, preferably TNF-α, as measured by standard binding assays as arewell known in the art (See, e.g., U.S. Pat. No. 5,945,397 to Smith,cols. 16-17). Preferably, receptors of the present invention are capableof binding greater than 0.1 nmoles TNF-α/nmole receptor, and morepreferably, greater than 0.5 nmoles TNF-α/nmole receptor using standardbinding assays.

As used herein, the term “regulatory element” refers to a nucleotidesequence involved in an interaction of molecules that contributes to thefunctional regulation of a nucleic acid, including but not limited to,replication, duplication, transcription, splicing, translation, ordegradation of the nucleic acid. The regulation may be enhancing orinhibitory in nature. Regulatory elements known in the art include, forexample, transcriptional regulatory sequences such as promoters andenhancers. A promoter is a DNA region that is capable under certainconditions of aiding the initiation of transcription of a coding regionusually located downstream (in the 3′ direction) from the promoter.

As used herein, the term “operably linked” refers to a juxtaposition ofgenetic elements, wherein the elements are in a relationship permittingthem to operate in the expected manner. For example, a promoter isoperably linked to a coding region if the promoter helps initiatetranscription of the coding sequence. As long as this functionalrelationship is maintained, there can be intervening residues betweenthe promoter and the coding region.

As used herein, the terms “transformation” or “transfection” refer tothe insertion of an exogenous nucleic acid into a cell, irrespective ofthe method used for the insertion, for example, lipofection,transduction, infection or electroporation. The exogenous nucleic acidcan be maintained as a non-integrated vector, for example, a plasmid, oralternatively, can be integrated into the cell's genome.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids. or viralvectors (e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses).

As used herein, the term “isolated protein” refers to a protein orpolypeptide that is not naturally-occurring and/or is separated from oneor more components that are naturally associated with it.

As used herein, the term “isolated nucleic acid” refers to a nucleicacid that is not naturally-occurring and/or is in the form of a separatefragment or as a component of a larger construct, which has been derivedfrom a nucleic acid isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials, and in a quantity orconcentration enabling identification and manipulation by standardbiochemical methods, for example, using a cloning vector.

As used herein the term “purified protein” refers to a protein that ispresent in the substantial absence of other protein. However, suchpurified proteins can contain other proteins added as stabilizers,carriers, excipients, or co-therapeutics. The Lena “purified” as usedherein preferably means at least 80% by dry weight, more preferably inthe range of 95-99% by weight, and most preferably at least 99.8% byweight, of protein present, excluding proteins added as stabilizers,carriers, excipients, or co-therapeutics.

As used herein, the term “altering the splicing of a pre-mRNA” refers toaltering the splicing of a cellular pre-mRNA target resulting in analtered ratio of splice products. Such an alteration of splicing can bedetected by a variety of techniques well known to one of skill in theart. For example, RT-PCR on total cellular RNA can be used to detect theratio of splice products in the presence and the absence of an SSO.

As used herein, the term “complementary” is used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between an oligonucleotide and a DNA or RNAcontaining the target sequence. It is understood in the art that thesequence of an oligonucleotide need not be 100% complementary to that ofits target. For example, for an SSO there is a sufficient degree ofcomplementarity when, under conditions which permit splicing, binding tothe target will occur and non-specific binding will be avoided.

Proteins:

One embodiment of the present invention is a protein, either full lengthor mature, which is encoded by a cDNA derived from a mammalian TNFRgene, and in the cDNA exon 6 is followed directly by exon 8 and as aresult lacks exon 7. Furthermore the protein can bind TNF, preferablyTNF-α, and can act as a TNF, preferably TNF-α, antagonist. Preferably,TNFR of the present invention is capable of inhibition of TNF-mediatedcytotoxicity to a greater extent than the soluble extracellular domainalone, and more preferably, to an extent comparable to or greater thanTNFR:Fc. Mammalian TNFR according to the present disclosure includes,but is not limited to, human, primate, murine, canine, feline, bovine,ovine, equine, and porcine TNFR. Furthermore, mammalian TNFR accordingto the present disclosure includes, but is not limited to, a proteinsequence that results from one or more single nucleotide polymorphisms,such as for example those disclosed in EP Pat. Appl. 1,172,444, as longas the protein retains a comparable biological activity to the referencesequence with which it is being compared.

In one embodiment, the mammalian TNFR is a mammalian TNFR1, preferably ahuman TNFR1. For human TNFR1 two non-limiting examples of thisembodiment are given by huTNFR1 Δ7 which includes the signal sequence asshown in SEQ ID No: 6 and mature huTNFR1 Δ7 (amino acids 30-417 of SEQID No: 6) which lacks the signal sequence. The sequences of thesehuTNFR1 Δ7 proteins are either amino acids 1-208 of wild type humanTNFR1 (SEQ ID No: 2) which includes the signal sequence or 30-208 ofwild type human TNFR1 for mature huTNFR1 Δ7 which lacks the signalsequence, and in either case is followed immediately by amino acids247-455 of wild type human TNFR1.

In another preferred embodiment, the mammalian TNFR is a mammalianTNFR2, most preferably a human TNFR2. For human TNFR2 two non-limitingexamples of this embodiment are given by huTNFR2 Δ7 which includes thesignal sequence as shown in SEQ ID No: 10 or mature huTNFR2 Δ7 (aminoacids 23-435 of SEQ ID No: 10) which lacks the signal sequence. Thesequences of these huTNFR2 Δ7 proteins are either amino acids 1-262 ofwild type human TNFR2 (SEQ ID No: 4) which includes the signal sequenceor 23-262 of wild type human TNFR2 for mature huTNFR2 Δ7 which lacks thesignal sequence, followed in either case by the amino acid glutamate,because of the creation of a unique codon at the exon 6-8 junction,which is followed by amino acids 290-461 of wild type human TNFR2.

The proteins of the present invention also include those proteins thatare chemically modified. Chemical modification of a protein refers to aprotein where at least one of its amino acid residues is modified byeither natural processes, such as processing or other post-translationalmodifications, or by chemical modification techniques known in the art.Such modifications include, but are not limited to, acetylation,acylation, amidation, ADP-ribosylation, glycosylation, methylation,pegylation, prenylation, phosphorylation, or cholesterol conjugation.

Nucleic Acids:

One embodiment of the present invention is a nucleic acid that encodes aprotein, either full length or mature, which is encoded by a cDNAderived from a mammalian TNFR gene, and in the cDNA exon 6 is followeddirectly by exon 8 and as a result lacks exon 7.

Such sequences are preferably provided in the form of an open readingframe uninterrupted by internal nontranslated sequences, or introns,which are typically present in eukaryotic genes. Genomic DNA containingthe relevant sequences can also be used. In one embodiment, the nucleicacid is either an mRNA or a cDNA. In another embodiment, it is genomicDNA.

In one embodiment, the mammalian TNFR is a mammalian TNFR1. For thisembodiment, the mammalian TNFR1 is preferably a human TNFR1. For humanTNFR1, two non-limiting examples of this embodiment are nucleic acidswhich encode the huTNFR1 Δ7 which includes the signal sequence as shownin SEQ ID No: 6 and mature huTNFR1 Δ7 (amino acids 30-417 of SEQ ID No:6) which lacks the signal sequence. Preferably, the sequences of thesehuTNFR1 Δ7 nucleic acids are nucleotides 1-1251 of SEQ ID No: 5, whichincludes the signal sequence and nucleotides 88-1251 of SEQ ID No: 5which lacks the signal sequence. The sequences of these huTNFR1 Δ7nucleic acids are either nucleotides 1-625 of wild type human TNFR1 (SEQID No: 1) which includes the signal sequence or 88-625 of wild typehuman TNFR1 for mature huTNFR2 Δ7 which lacks the signal sequence, andin either case is followed immediately by amino acids 740-1368 of wildtype human TNFR1.

In another preferred embodiment, the mammalian TNFR is a mammalianTNFR2, most preferably a human TNFR2. For, human TNFR2, two non-limitingexamples of this embodiment are nucleic acids which encode the huTNFR2Δ7 which includes the signal sequence as shown in SEQ ID No: 10 ormature huTNFR2 Δ7 (amino acids 23-435 of SEQ ID No: 10) which lacks thesignal sequence. Preferably, the sequences of these huTNFR2 Δ7 nucleicacids are nucleotides 1-1305 of SEQ ID No: 9 which includes the signalsequence and nucleotides 67-1305 of SEQ ID No: 9 which lacks the signalsequence. The sequences of these huTNFR2 Δ7 nucleic acids are eithernucleotides 1-787 of wild type human TNFR2 (SEQ ID No: 3) which includesthe signal sequence or 67-787 of wild type human TNFR2 for maturehuTNFR2 Δ7 which lacks the signal sequence, and in either case isfollowed immediately by amino acids 866-1386 of wild type human TNFR2.

The bases of the nucleic acids of the present invention can be theconventional bases cytosine, guanine, adenine and uracil or thymidine.Alternatively, modified bases can be used. Other suitable bases include,but are not limited to, 5-methylcytosine (^(Me)C), isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propyny-6,5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine,2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine,2-chloro-6-aminopurine and 9-(aminoethoxy)phenoxazine.

Suitable nucleic acids of the present invention include numerousalternative chemistries. For example, suitable nucleic acids of thepresent invention include, but are not limited to, those wherein atleast one of the internucleotide bridging phosphate residues is amodified phosphate, such as phosphorothioate, methyl phosphonate, methylphosphonothioate, phosphoromorpholidate, phosphoropiperazidate, andphosphoroamidate. In another non-limiting example, suitable nucleicacids of the present invention include those wherein at least one of thenucleotides contain a 2′ lower alkyl moiety (e.g., C₁-C₄, linear orbranched, saturated or unsaturated alkyl, such as methyl, ethyl,ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).

Nucleic acids of the present invention also include, but are not limitedto, those wherein at least one, of the nucleotides is a nucleic acidanalogue. Examples of such analogues include, but are not limited to,hexitol (HNA) nucleotides, 2′O-4′C-linked bicyclic ribofuranosyl (LNA)nucleotides, peptide nucleic acid (PNA) analogues, N3′→P5′phosphoramidate analogues, phosphorodiamidate morpholino nucleotideanalogues, and combinations thereof.

Nucleic acids of the present invention include, but are not limited to,modifications of the nucleic acids involving chemically linking to thenucleic acids one or more moieties or conjugates. Such moieties include,but are not limited to, lipid moieties such as a cholesterol moiety,cholic acid, a thioether, e.g. hexyl-S-tritylthiol, a thiocholesterol,an aliphatic chain, e.g., dodecandiol or undecyl residues, aphospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

Pharmaceutical Compositions and Preparations:

Other embodiments of the invention are pharmaceutical compositionscomprising the foregoing proteins and nucleic acids.

The nucleic acids and proteins of the present invention may be admixed,encapsulated, conjugated, or otherwise associated with other molecules,molecule structures, or mixtures of compounds, as for example liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution, and/or absorption.

Formulations of the present invention comprise nucleic acids andproteins in a physiologically or pharmaceutically acceptable carrier,such as an aqueous carrier. Thus formulations for use in the presentinvention include, but are not limited to, those suitable for parenteraladministration including intra-articular, intraperitoneal, intravenous,intraarterial, subcutaneous, or intramuscular injection or infusion, aswell as those suitable for topical, ophthalmic, vaginal, oral, rectal orpulmonary administration (including inhalation or insufflation ofpowders or aerosols, including by nebulizer, intratracheal, andintranasal delivery). The formulations may conveniently be presented inunit dosage form and may be prepared by any of the methods well known inthe art. The most suitable route of administration in any given case maydepend upon the subject, the nature and severity of the condition beingtreated, and the particular active compound which is being used.

Pharmaceutical compositions of the present invention include, but arenot limited to, physiologically and pharmaceutically acceptable salts,i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological properties. Examplesof such salts are (a) salts formed with cations such as sodium,potassium, NH₄ ⁺, magnesium, calcium, polyamines such as spermine andspermidine, etc.; (b) acid addition salts formed with inorganic acids,for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and (c) salts formed withorganic acids such as, for example, acetic acid, oxalic acid, tartaricacid, succinic acid, maleic acid, fumaric acid, gluconic acid, citricacid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmiticacid, alginic acid, polyglutamic acid, napthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, napthalenedisulfonic acid,polygalacturonic acid, and the like.

The present invention provides for the use of proteins and nucleic acidsas set forth above for the preparation of a medicament for treating apatient afflicted with an inflammatory disorder involving excessiveactivity of TNF, as discussed below. In the manufacture of a medicamentaccording to the invention, the nucleic acids and proteins of thepresent invention are typically admixed with, inter alia, an acceptablecarrier. The carrier must, of course, be acceptable in the sense ofbeing compatible with other ingredients in the formulation and must notbe deleterious to the patient. The carrier may be a solid or liquid.Nucleic acids and proteins of the present invention are incorporated informulations, which may be prepared by any of the well known techniquesof pharmacy consisting essentially of admixing the components,optionally including one or more accessory therapeutic ingredients.

Formulations of the present invention may comprise sterile aqueous andnon-aqueous injection solutions of the active compounds, whichpreparations are preferably isotonic with the blood of the intendedrecipient and essentially pyrogen free. These preparations may containanti-oxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions can include, but are not limited to,suspending agents and thickening agents. The formulations may bepresented in unit dose or multi-dose containers, for example, sealedampoules and vials, and may be stored in freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.

In the formulation the nucleic acids and proteins of the presentinvention may be contained within a particle or vesicle, such as aliposome or microcrystal, which may be suitable for parenteraladministration. The particles may be of any suitable structure, such asunilamellar or plurilameller, so long as the nucleic acids and proteinsof the present invention are contained therein. Positively chargedlipids such asN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummethylsulfate, or“DOTAP,” are particularly preferred for such particles and vesicles. Thepreparation of such lipid particles is well known (See references inU.S. Pat. No. 5,976,879 col. 6).

Expression Vectors and Host Cells:

The present invention provides expression vectors to amplify or expressDNA encoding mammalian TNFR of the current invention. The presentinvention also provides host cells transformed with the foregoingexpression vectors. Expression vectors are replicable DNA constructswhich have synthetic or cDNA-derived DNA fragments encoding mammalianTNFR or bioequivalent analogues operably linked to suitabletranscriptional or translational regulatory elements derived frommammalian, microbial, viral, or insect genes. A transcriptional unitgenerally comprises an assembly of (a) a genetic element or elementshaving a regulatory role in gene expression, such as, transcriptionalpromoters or enhancers, (b) a structural or coding sequence which istranscribed into mRNA and translated into protein, and (c) appropriatetranscription and translation initiation and termination sequences. Suchregulatory elements can include an operator sequence to controltranscription, and a sequence encoding suitable mRNA ribosomal bindingsites. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants, can additionally be incorporated.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA for a signal peptide (secretory leader) isoperably linked to DNA for a polypeptide if it is expressed as aprecursor which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, contiguous and in reading frame. Structuralelements intended for use in yeast expression systems preferably includea leader sequence enabling extracellular secretion of translated proteinby a host cell. Alternatively, where recombinant protein is expressedwithout a leader or transport sequence, it may include an N-terminalmethionine residue. This residue may optionally be subsequently cleavedfrom the expressed protein to provide a final product.

Mammalian TNFR DNA is expressed or amplified in a recombinant expressionsystem comprising a substantially homogeneous monoculture of suitablehost microorganisms, for example, bacteria such as E. coli or yeast suchas S. cerevisiae, which have stably integrated (by transformation ortransfection) a recombinant transcriptional unit into chromosomal DNA orcarry the recombinant transcriptional unit as a component of a residentplasmid. Recombinant expression systems as defined herein will expressheterologous protein either constitutively or upon induction of theregulatory elements linked to the DNA sequence or synthetic gene to beexpressed.

Transformed host cells are cells which have been transformed ortransfected with mammalian TNFR vectors constructed using recombinantDNA techniques. Transformed host cells ordinarily express TNFR, but hostcells transformed for purposes' of cloning or amplifying TNFR DNA do notneed to express TNFR. Suitable host cells for expression of mammalianTNFR include prokaryotes, yeast, fungi, or higher eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include, but are notlimited to, established insect and mammalian cell lines. Cell-freetranslation systems can also be employed to produce mammalian TNFR usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are well known in the art.

Prokaryotic expression hosts may be used for expression of TNFR that donot require extensive proteolytic and disulfide processing. Prokaryoticexpression vectors generally comprise one or more phenotypic selectablemarkers, for example a gene encoding proteins conferring antibioticresistance or supplying an autotrophic requirement, and an origin ofreplication recognized by the host to ensure amplification within thehost. Suitable prokaryotic hosts for transformation include E. coli,Bacillus subtilis, Salmonella typhimurium, and various species withinthe genera Pseudomonas, Streptomyces, and Staphyolococcus, althoughothers can also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). These pBR322 “backbone” sections arecombined with an appropriate promoter and the structural sequence to beexpressed. pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells. Such commercial vectors include, for example, the series ofNovagen® pET vectors (EMD Biosciences, Inc., Madison, Wis.).

Promoters commonly used in recombinant microbial expression vectorsinclude the lactose promoter system, and the λ P_(L) promoter, the T7promoter, and the T7 lac promoter. A particularly useful bacterialexpression system, Novagen® pET system (EMD Biosciences, Inc., Madison,Wis.) employs a T7 or T7 lac promoter and E. coli strain, such asBL21(DE3) which contain a chromosomal copy of the T7 RNA polymerasegene.

TNFR proteins can also be expressed in yeast and fungal hosts,preferably from the genus Saccharomyces, such as S. cerevisiae. Yeast ofother genera, such as Pichia or Kluyveromyces can also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS),promoter, DNA encoding TNFR, sequences for polyadenylation andtranscription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerpermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 or URA3gene, which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan or uracil, respectively, and apromoter derived from a highly expressed yeast gene to inducetranscription of a structural sequence downstream. The presence of theTRP1 or URA3 lesion in the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan or uracil, respectively.

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are well known in the art.

Preferred yeast vectors can be assembled using DNA sequences from pUC18for selection and replication in E. coli (Amp′ gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The yeast α-factor leader,which directs secretion of heterologous proteins, can be insertedbetween the promoter and the structural gene to be expressed. The leadersequence can be modified to contain, near its 3′ end, one or more usefulrestriction sites to facilitate fusion of the leader sequence to foreigngenes. Suitable yeast transformation protocols are known to those ofskill in the art.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% or 4% glucose supplemented with 80 μg/ml adenine and 80μg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustionof medium glucose. Crude yeast supernatants are harvested by filtrationand held at 4° C. prior to further purification.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express TNFR protein. Expression of recombinant proteins inmammalian cells is particularly preferred because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, and other cell lines capable ofexpressing an appropriate vector including, for example, L cells, suchas L929, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK celllines. Mammalian expression vectors can comprise nontranscribed elementssuch as an origin of replication, a suitable promoter, for example, theCMVie promoter, the chicken beta-actin promoter, or the compositehEF1-HTLV promoter, and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking nontranscribed sequences, and 5′ or 3′nontranslated sequences, such as necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are known to thoseof skill in the art.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells can be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), humancytomegalovirus, such as the CMVie promoter, HTLV; such as the compositehEF1-HTLV promoter. DNA sequences derived from the SV40 viral genome,for example, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites can be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence.

Further, mammalian genomic TNFR promoter, such as control and/or signalsequences can be utilized, provided such control sequences arecompatible with the host cell chosen.

In preferred aspects of the present invention, recombinant expressionvectors comprising TNFR cDNAs are stably integrated into a host cell'sDNA.

Accordingly one embodiment of the invention is a method of treating aninflammatory disease or condition by administering a stable, secreted,ligand-binding form of a TNF receptor, thereby decreasing the activityof TNF for the receptor. In another embodiment, the invention is amethod of treating an inflammatory disease or condition by administeringan oligonucleotide that encodes a stable, secreted, ligand-binding formof a TNF receptor, thereby decreasing the activity of TNF for thereceptor. In another embodiment, the invention is a method of producinga stable, secreted, ligand-binding form of a TNF receptor.

The following aspects of the present invention discussed below apply tothe foregoing embodiments.

The methods, nucleic acids, proteins, and formulations of the presentinvention are also useful as in vitro or in vivo tools.

Embodiments of the invention can be used to treat any condition in whichthe medical practitioner intends to limit the effect of TNF or asignalling pathway activated by it. In particular, the invention can beused to treat an inflammatory disease. In one embodiment, the conditionis an inflammatory systemic disease, e.g., rheumatoid arthritis orpsoriatic arthritis. In another embodiment, the disease is aninflammatory liver disease. Examples of inflammatory liver diseasesinclude, but are not limited to, hepatitis associated with the hepatitisA, B, or C viruses, alcoholic liver disease, and non-alcoholicsteatosis. In yet another embodiment, the inflammatory disease is a skincondition such as psoriasis.

The uses of the present invention include, but are not limited to,treatment of diseases for which known TNF antagonists have been shownuseful. Three specific TNF antagonists are currently FDA-approved. Thedrugs are etanercept (Enbrel®), infliximab (Remicade®) and adalimumab(Humira®). One or more of these drugs is approved for the treatment ofrheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis,psoriatic arthritis, ankylosing spondylitis, and inflammatory boweldisease (Crohn's disease or ulcerative colitis).

Protein Expression and Purification:

When mammalian or insect cells are used, properly expressed TNFR proteinwill be secreted into the extracellular media. The protein is recoveredfrom the media, and is concentrated and is purified using standardbiochemical techniques. After expression in mammalian cells bylentiviral or AAV transduction, plasmid transfection, or any similarprocedure, or in insect cells after baculoviral transduction, theextracellular media of these cells is concentrated using concentrationfilters with an appropriate molecular weight cutoff, such as Amicon®filtration units. To avoid loss of TNFR protein, the filter should allowproteins to flow through that are at or below 50 kDal.

When TNFR protein is expressed in bacterial culture it can be purifiedby standard biochemical techniques. Bacteria are lysed, and the cellularextract containing the TNFR is desalted and is concentrated.

In either case, the TNFR protein is preferably purified by affinitychromatography. The use of column chromatography with an affinity matrixcomprising TNF-α is preferred. Alternatively, an affinity purificationtag can be added to either the N- or the C-terminus of the TNFR protein.For example, a polyhistidine-tag (His-tag), which is an amino acid motifwith at least six histidines, can be used for this purpose (Hengen, P.,1995, Trends Biochem. Sci. 20:285-86). The addition of a His-tag can beachieved by the in-frame addition of a nucleotide sequence encoding theHis-tag directly to either the 5′ or 3′ end of the TNFR open readingframe in an expression vector. One such nucleotide sequence for theaddition of a C-terminal His-tag is given in SEQ ID No: 126. When aHis-tag is incorporated into the protein, a nickel or cobalt affinitycolumn is employed to purify the tagged TNFR, and the His-tag canoptionally then be cleaved. Other suitable affinity purification tagsand methods of purification of proteins with those tags are well knownin the art.

Alternatively, a non-affinity based purification scheme can be used,involving fractionation of the TNFR extracts on a series of columns thatseparate the protein based on size (size exclusion chromatography),charge (anion and cation exchange chromatography) and hydrophobicity(reverse phase chromatography). High performance liquid chromatographycan be used to facilitate these steps.

Other methods for the expression and purification of TNFR proteins arewell known (See, e.g., U.S. Pat. No. 5,605,690 to Jacobs).

Use of Proteins for the Treatment of Inflammatory Diseases:

For therapeutic use, purified TNFR proteins of the present invention areadministered to a patient, preferably a human, for treatingTNF-dependent inflammatory diseases, such as arthritis. In the treatmentof humans, the use of huTNFRs is preferred. The TNFR proteins of thepresent invention can be administered by bolus injection, continuousinfusion, sustained release from implants, or other suitable techniques.Typically, TNFR therapeutic proteins will be administered in the form ofa composition comprising purified protein in conjunction withphysiologically acceptable carriers, excipients or diluents. Suchcarriers will be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the TNFR with buffers, antioxidants suchas ascorbic acid, polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with conspecific serum albumin are exemplaryappropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions, for example,sucrose, as diluents. Preservatives, such as benzyl alcohol may also beadded. The amount and frequency of administration will depend of course,on such factors as the nature and the severity of the indication beingtreated, the desired response, the condition of the patient and soforth.

TNFR proteins of the present invention are administered systemically intherapeutically effective amounts preferably ranging from about 0.1mg/kg/week to about 100 mg/kg/week. In preferred embodiments, TNFR isadministered in amounts ranging from about 0.5 mg/kg/week to about 50mg/kg/week. For local administration, dosages preferably range fromabout 0.01 mg/kg to about 1.0 mg/kg per injection.

Use of Expression Vectors to Increase the Levels of a TNF Antagonist ina Mammal:

The present invention provides a process of increasing the levels of aTNF antagonist in a mammal. The process includes the step oftransforming cells of the mammal with an expression vector describedherein, which drives expression of a TNFR as described herein.

The process is particularly useful in large mammals such as domesticpets, those used for food production, and primates. Exemplary largemammals are dogs, cats, horses cows, sheep, deer, and pigs. Exemplaryprimates are monkeys, apes, and humans.

The mammalian cells can be transformed either in vivo or ex vivo. Whentransformed in vivo, the expression vector are administered directly tothe mammal, such as by injection. Means for transforming cells in vivoare well known in the art. When transformed ex vivo, cells are removedfrom the mammal, transformed ex vivo, and the transformed cells arereimplanted into the mammal.

Splice-Switching Oligomers (SSOs):

In another aspect, the present invention employs splice switchingoligonucleotides or splice switching oligomers (SSOs) to control thealternative splicing of TNFR2 so that the amount of a soluble,ligand-binding form that lacks exon 7 is increased and the amount of theintegral membrane form is decreased. The methods and compositions of thepresent invention can be used in the treatment of diseases associatedwith excessive TNF activity.

Accordingly, one embodiment of the invention is a method of treating aninflammatory disease or condition by administering SSOs to a patient.The SSOs that are administered alter the splicing of a pre-mRNA toproduce a mammalian TNFR2 protein that lacks exon 7, In anotherembodiment, the invention is a method of producing a mammalian TNFR2protein that lacks exon 7 in a cell by administering SSOs to the cell.

The length of the SSO (i.e. the number of monomers in the oligomer) issimilar to an antisense oligonucleotide (ASON), typically between about8 and 30 nucleotides. In preferred embodiments, the SSO will be betweenabout 10 to 16 nucleotides. The invention can be practiced with SSOs ofseveral chemistries that hybridize to RNA, but that do not activate thedestruction of the RNA by RNase H, as do conventional antisense 2′-deoxyoligonucleotides. The invention can be practiced using 2′O modifiednucleic acid oligomers, such as where the 2′O is replaced with —O—CH₃,—O—CH₂—CH₂—O—CH₃, —O—CH₂—CH₂—CH₂—NH₂, —O—CH₂—CH₂—CH₂—OH or —F, where2′O-methyl or 2′O-methyloxyethyl is preferred. The nucteobases do notneed to be linked to sugars; so-called peptide nucleic acid oligomers ormorpholine-based oligomers can be used. A comparison of these differentlinking chemistries is found in Sazani, P. et al., 2001, Nucleic AcidsRes. 29:3695. The term splice-switching oligonucleotide is intended tocover the above forms. Those skilled in the art will appreciate therelationship between antisense oligonucleotide gapmers and SSOs. Gapmersare ASON that contain an RNase H activating region (typically a2′-deoxyribonucleoside phosphorothioate) which is flanked bynon-activating nuclease resistant oligomers. In general, any chemistrysuitable for the flanking sequences in a gapmer ASON can be used in anSSO.

The SSOs of this invention may be made through the well-known techniqueof solid phase synthesis. Any other means for such synthesis known inthe art may additionally or alternatively be used. It is well known touse similar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The bases of the SSO may be the conventional cytosine, guanine, adenineand uracil or thymidine. Alternatively, modified bases can be used. Ofparticular interest are modified bases that increase binding affinity.One non-limiting example of preferred modified bases are the so-calledG-clamp or 9-(aminoethoxy)phenoxazine nucleotides, cytosine analoguesthat form 4 hydrogen bonds with guanosine. (Flanagan, W. M., et al.,1999, Proc. Natl. Acad. Sci. 96:3513; Holmes, S. C., 2003, Nucleic AcidsRes. 31:2759). Specific examples of other bases include, but are notlimited to, 5-methylcytosine (^(Me)C), isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 5-propyny-6, 5-methylthiazoleuracil,6-aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine,7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine and2-chloro-6-aminopurine.

A particularly preferred chemistry is provided by locked nucleic acids(LNA) (Koshkin, A. A., et al., 1998, Tetrahedron 54:3607; Obika, S., etal., 1998, Tetrahedron Left. 39:5401). As used herein, the terms “LNAunit”, “LNA monomer”, “LNA residue”, “locked nucleic acid unit”, “lockednucleic acid monomer” or “locked nucleic acid residue”, refer to abicyclic nucleoside analogue. LNA units and methods of their synthesisare described in inter alia WO 99/14226, WO 00/56746, WO 00/56748, WO01/25248, WO 02/28875, WO 03/006475 and WO 03/095467. The LNA unit mayalso be defined with respect to its chemical formula. Thus, an “LNAunit”, as used herein, has the chemical structure shown in Formula 1below:

wherein,

X is selected from the group consisting of O, S and NRH, where R is H orC₁-C₄-alkyl;

Y is (—CH₂)_(r), where r is an integer of 1-4; and

B is a base of natural or non-natural origin as described above.

In a preferred embodiment, r is 1 or 2, and in a more preferredembodiment r is 1.

When LNA nucleotides are employed in an SSO it is preferred that non-LNAnucleotides also be present. LNA nucleotides have such high affinitiesof hybridization that there can be significant non-specific binding,which may reduce the effective concentration of the free-SSO. When LNAnucleotides are used they may be alternated conveniently with2′-deoxynucleotides. The pattern of alternation is not critical.Alternating nucleotides, alternating dinucleotides or mixed patterns,e.g., LDLDLD or LLDLLD or LDDLDD can be used. For example in oneembodiment, contains a sequence of nucleotides selected from the groupconsisting of: LdLddLLddLdLdLL, LdLdLLLddLLLdLL, LMLMMLLMMLMLMLL,LMLMLLLMMLLLMLL, LFLFFLLFFLFLFLL, LFLFLLLFFLLLFLL, LddLddLddL,dLddLddLdd, ddLddLddLd, LMMLMMLMML, MLMMLMMLMM, MMLMMLMMLM, LFFLFFLFFL,FLFFIFFLFF, FFLFFLFFLF, dLdLdLdLdL, LdLdLdLdL, MLMLMLMLML, LMLMLMLML,FLFLFLFLFL, LFLFLFLFL, where L is a LNA unit, d is a DNA unit, M is2′MOE, F is 2′Fluoro.

When 2′-deoxynucleotides or 2′-deoxynucleoside phosphorothioates aremixed with LNA nucleotides it is important to avoid RNase H activation.It is expected that between about one third and two thirds of the LNAnucleotides of an SSO will be suitable. When affinity-enhancingmodifications are used, including but not limited to LNA or G-clampnucleotides, the skilled person recognizes it can be necessary toincrease the proportion of such affinity-enhancing modifications.

Numerous alternative chemistries which do not activate RNase H areavailable. For example, suitable SSOs can be oligonucleotides wherein atleast one of the internucleotide bridging phosphate residues is amodified phosphate, such as methyl phosphonate, methyl phosphonothioate,phosphoromorpholidate, phosphoropiperazidate, and phosphoroamidate. Forexample, every other one of the internucleotide bridging phosphateresidues may be modified as described. In another non-limiting example,such SSO are oligonucleotides wherein at least one of the nucleotidescontains a 2′ lower alkyl moiety (e.g., C₁-C₄, linear or branched,saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl,1-propenyl, 2-propenyl, and isopropyl). For example, every other one ofthe nucleotides may be modified as described. (See references in U.S.Pat. No. 5,976,879 col. 4). For in vivo use, phosphorothioate linkagesare preferred.

The length of the SSO will be from about 8 to about 30 bases in length.Those skilled in the art appreciate that when affinity-increasingchemical modifications are used, the SSO can be shorter and still retainspecificity. Those skilled in the art will further appreciate that anupper limit on the size of the SSO is imposed by the need to maintainspecific recognition of the target sequence, and to avoidsecondary-structure forming self hybridization of the SSO and by thelimitations of gaining cell entry. These limitations imply that an SSOof increasing length (above and beyond a certain length which willdepend on the affinity of the SSO) will be more frequently found to beless specific, inactive or poorly active.

SSOs of the invention include, but are not limited to, modifications ofthe SSO involving chemically linking to the SSO one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the SSO. Such moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipids, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

It is not necessary for all positions in a given SSO to be uniformlymodified, and in fact more than one of the aforementioned modificationsmay be incorporated in a single compound or even at a single nucleosidewithin an SSO.

The SSOs may be admixed, encapsulated, conjugated, or otherwiseassociated with other molecules, molecule structures, or mixtures ofcompounds, as for example liposomes, receptor targeted molecules, oral,rectal, topical or other formulation, for assisting in uptake,distribution, and/or absorption.

Those skilled in the art appreciate that cellular differentiationincludes, but is not limited to, differentiation of the spliceosome.Accordingly, the activity of any particular SSO can depend upon the celltype into which they are introduced. For example, SSOs which areeffective in one cell type may be ineffective in another cell type.

The methods, oligonucleotides, and formulations of the present inventionare also useful as in vitro or in vivo tools to examine splicing inhuman or animal genes. Such methods can be carried out by the proceduresdescribed herein, or modifications thereof which will be apparent toskilled persons.

The SSOs disclosed herein can be used to treat any condition in whichthe medical practitioner intends to limit the effect of TNF or thesignalling pathway activated by TNF. In particular, the invention can beused to treat an inflammatory disease. In one embodiment, the conditionis an inflammatory systemic disease, e.g., rheumatoid arthritis orpsoriatic arthritis. In another embodiment, the disease is aninflammatory liver disease. Examples of inflammatory liver diseasesinclude, but are not limited to, hepatitis associated with the hepatitisA, B, or C viruses, alcoholic liver disease, and non-alcoholicsteatosis. In yet another embodiment, the inflammatory disease is a skincondition such as psoriasis.

The uses of the present invention include, but are not limited to,treatment of diseases for which known TNF antagonists have been shownuseful. Three specific TNF antagonists are currently FDA-approved. Thedrugs are etanercept (Enbrel®), infliximab (Remicade®) and adalimumab(Hurnira®). One or more of these drugs is approved for the treatment ofrheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis,psoriatic arthritis, ankylosing spondylitis, and inflammatory boweldisease (Crohn's disease or ulcerative colitis).

The administration of the SSO to subjects can be accomplished usingprocedures developed for ASON. ASON have been successfully administeredto experimental animals and human subjects by intravenous administrationin saline in doses as high as 6 mg/kg three times a week (Yacysyhn, etal., 2002, Gut 51:30 (anti-ICAM-1 ASON for treatment of Crohn'sdisease); Stevenson, J., et al., 1999, J. Clinical Oncology 17:2227(anti-RAF-1 ASON targeted to PBMC)). The pharmacokinetics of 2′O-MOEphosphorothioate ASON, directed towards TNF-α has been reported (Geary,R. S., et al., 2003, Drug Metabolism and Disposition 31:1419). Thesystemic efficacy of mixed LNA/DNA molecules has also been reported(Fluiter, K., et al., 2003, Nucleic Acids Res. 31:953).

The systemic activity of SSO in a mouse model system was investigatedusing 2′O-MOE phosphorothioates and PNA chemistries. Significantactivity was observed in all tissues investigated except brain, stomachand dermis (Sazani, P., et al., 2002, Nature Biotechnology 20, 1228).

In general any method of administration that is useful in conventionalantisense treatments can be used to administer the SSO of the invention.For testing of the SSO in cultured cells, any of the techniques thathave been developed to test ASON or SSO may be used.

Formulations of the present invention comprise SSOs in a physiologicallyor pharmaceutically acceptable carrier, such as an aqueous carrier. Thusformulations for use in the present invention include, but are notlimited to, those suitable for parenteral administration includingintraperitoneal, intraarticular, intravenous, intraarterial,subcutaneous, or intramuscular injection or infusion, as well as thosesuitable for topical, ophthalmic, vaginal, oral, rectal or pulmonary(including inhalation or insufflation of powders or aerosols, includingby nebulizer, intratracheal, intranasal delivery) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art. The mostsuitable route of administration in any given case may depend upon thesubject, the nature and severity of the condition being treated, and theparticular active compound which is being used.

Pharmaceutical compositions of the present invention include, but arenot limited to, physiologically and pharmaceutically acceptable salts,i.e, salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological properties. Examplesof such salts are (a) salts formed with cations such as sodium,potassium, NH₄ ⁺, magnesium, calcium, polyamines such as spermine andspermidine, etc.; (b) acid addition salts formed with inorganic acids,for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and (c) salts formed withorganic acids such as, for example, acetic acid, oxalic acid, tartaricacid, succinic acid, maleic acid, fumaric acid, gluconic acid, citricacid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmiticacid, alginic acid, polyglutamic acid, napthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, napthalenedisulfonic acid,polygalacturonic acid, and the like.

The present invention provides for the use of SSOs having thecharacteristics set forth above for the preparation of a medicament forincreasing the ratio of a mammalian TNFR2 protein that lacks exon 7 toits corresponding membrane bound form, in a patient afflicted with aninflammatory disorder involving TNF-α, as discussed above. In themanufacture of a medicament according to the invention, the SSOs aretypically admixed with, inter alia, an acceptable carrier. The carriermust, of course, be acceptable in the sense of being compatible with anyother ingredients in the formulation and must not be deleterious to thepatient. The carrier may be a solid or liquid. SSOs are incorporated inthe formulations of the invention, which may be prepared by any of thewell known techniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory therapeuticingredients.

Formulations of the present invention may comprise sterile aqueous andnon-aqueous injection solutions of the active compounds, whichpreparations are preferably isotonic with the blood of the intendedrecipient and essentially pyrogen free. These preparations may containanti-oxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions can include, but are not limited to,suspending agents and thickening agents. The formulations may bepresented in unit dose or multi-dose containers, for example, sealedampoules and vials, and may be stored in freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.

In the formulation the SSOs may be contained within a particle orvesicle, such as a liposome, or microcrystal, which may be suitable forparenteral administration. The particles may be of any suitablestructure, such as unilamellar or plurilameller, so long as the SSOs arecontained therein. Positively charged lipids such asN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummethylsulfate, or“DOTAP,” are particularly preferred for such particles and vesicles. Thepreparation of such lipid particles is well known. [See references inU.S. Pat. No. 5,976,879 col. 6]

The SSO can be targeted to any element or combination of elements thatregulate splicing, including the 3′splice site, the 5′ splice site, thebranch point, the polypyrimidine tract, exonic splicing ehancers, exonicsplicing silencers, intronic splicing enhancers, and intronic splicingsilencers.

Those skilled in the art can appreciate that the invention as directedtoward human TNFR2 can be practiced using SSO having a sequence that iscomplementary to at least 8, to at least 9, to at least 10, to at least11, to at least 12, to at least 13, to at least 14, to at least 15,preferably between 10 and 16 nucleotides of the portions of the TNFR2gene comprising exons 7 and its adjacent introns. SEQ ID No: 13 containsthe sequence of exon 7 of TNFR2 and 50 adjacent nucleotides of theflanking introns. For example, SSO targeted to human TNFR2 can have asequence selected from the sequences listed in Table 1. Whenaffinity-enhancing modifications are used, including but not limited toLNA or G-clamp nucleotides, the skilled person recognizes the length ofthe SSO can be correspondingly reduced. The pattern of alternation ofLNA and conventional nucleotides is not important.

TABLE 1 SSOs Targeted to Human TNFR2 SEQ ID. Name Sequence 5′ to 3′ 143378 CCA CAA TCA GTC CTA G 15 SK101   A CAA TCA GTC CTA G 16 SK102     AA TCA GTC CTA G 17 SK103         TCA GTC CTA G 18 SK104CCA CAA TCA GTC CT 19 SK105 CCA CAA TCA GTC 20 SK106 CCA CAA TCA G 21SK107  CA CAA TCA GTC CTA 22 SK108  CA CAA TCA GTC C 23 SK109  A CAA TCA GTC CT 24 SK110     CAA TCA GTC CTA 25 SK111  CA CAA TCA GT26 SK112   A CAA TCA GTC 27 SK113     CAA TCA GTC C 28 SK114     AA TCA GTC CT 29 SK115       A TCA GTC CTA 30 3379CAG TCC TAG AAA GAA A 31 SK117   G TCC TAG AAA GAA A 32 SK118     CC TAG AAA GAA A 33 SK119         TAG AAA GAA A 34 SK120CAG TCC TAG AAA GA 35 SK121 CAG TCC TAG AAA 36 SK122 CAG TCC TAG A 37SK123  AG TCC TAG AAA GAA 38 SK124  AG TCC TAG AAA G 39 SK125  G TCC TAG AAA GA 40 SK126     TCC TAG AAA GAA 41 SK127  AG TCC TAG AA42 SK128   G TCC TAG AAA 43 SK129     TCC TAG AAA G 44 SK130     CC TAG AAA GA 45 SK131       C TAG AAA GAA 46 3384ACT TTT CAC CTG GGT C 47 SK133   T TTT CAC CTG GGT C 48 SK134     TT CAC CTG GGT C 49 SK135         CAC CTG GGT C 50 SK136ACT TTT CAC CTG GG 51 SK137 ACT TTT CAC CTG 52 SK138 ACT TTT CAC C 53SK139  CT TTT CAC CTG GGT 54 SK140  CT TTT CAC CTG G 55 SK141  T TTT CAC CTG GG 56 SK142     TTT CAC CTG GGT 57 SK143  CT TTT CAC CT58 SK144   T TTT CAC CTG 59 SK145     TTT CAC CTG G 60 SK146     TT CAC CTG GG 61 SK147       T CAC CTG GGT

Those skilled in the art will also recognize that the selection of SSOsequences must be made with care to avoid a self-complementary SSO,which may lead to the formation of partial “hairpin” duplex structures.In addition, high GC content should be avoided to minimize thepossibility of non-specific base pairing. Furthermore, SSOs matchingoff-target genes, as revealed for example by BLAST, should also beavoided.

In some situations, it may be preferred to select an SSO sequence thatcan target a human and at least one other species. These SSOs can beused to test and to optimize the invention in said other species beforebeing used in humans, thereby being useful for regulatory approval anddrug development purposes. For example, SSOs with sequences selectedfrom SEQ ID Nos: 14, 30, 46, 70 and 71 which target human TNFR2 are also100% complementary to the corresponding Macaca Mullata sequences. As aresult these sequences can be used to test treatments in monkeys, beforebeing used in humans.

It will be appreciated by those skilled in the art that variousomissions, additions and modifications may be made to the inventiondescribed above without departing from the scope of the invention, andall such modifications and changes are intended to fall within the scopeof the invention, as defined by the appended claims. All sequencecitations, references, patents, patent applications or other documentscited referred to herein are incorporated by reference.

Example 1 Materials and Methods

Oligonucleotides. Table 3 lists chimeric locked nucleic acid (LNA) SSOswith alternating 2′deoxy- and 2′O-4′-(methylene)-bicyclic-ribonucleosidephosphorothioates and having sequences as described in U.S. applicationSer. No. 11/595,485. These were synthesized by Santaris Pharma, Denmark.For each SSO, the 5′-terminal nucleoside was a2′O-4′-methylene-ribonucleoside and the 3′-terminal nucleoside was a2′deoxy-ribonucleoside. Table 4 shows the sequences of chimeric LNA SSOswith alternating 2′-O-methyl-ribonucleoside-phosphorothioates (2′-OMe)and 2′O-4′-(methylene)-bicyclic-ribonucleoside phosphorothioates. Thesewere synthesized by Santaris Pharma, Denmark. The LNA is shown incapital letters and the 2′-OME is shown in lower case letters.

Cell culture and transfections. L929 cells were maintained in minimalessential media supplemented with 10% fetal bovine serum and antibiotic(37° C., 5% CO₂). For transfection, L929 cells were seeded in 24-wellplates at 10⁵ cells per well and transfected 24 hrs later.Oligonucleotides were complexed, at the indicated concentrations, with 2μL of Lipofectamine™ 2000 transfection reagent (Invitrogen) as per themanufacturer's directions. The nucleotide/lipid complexes were thenapplied to the cells and incubated for 24 hrs. The media was thenaspirated and cells harvested with TRI-Reagent™ (MRC, Cincinnati, Ohio).

RT-PCR. Total RNA was isolated with TRI-Reagent (MRC, Cincinnati, Ohio)and TNFR1 or TNFR2 mRNA was amplified by GeneAmp® RT-PCR using rTthpolymerase (Applied Biosystems) following supplier directions.Approximately 200 ng of RNA was used per reaction. Primers used in theexamples described herein are included in Table 2. Cycles of PCRproceeded: 95° C., 60 sec; 56° C., 30 sec; 72° C., 60 sec for 22-30cycles total.

In some instances a Cy5-labeled dCTP (GE Healthcare) was included in thePCR step for visualization (0.1 μL per 50 μL PCR reaction). The PCRproducts were separated on a 10% non-denaturing polyacrylamide gel, andCy5-labeled bands were visualized with a Typhoon™ 9400 Scanner (GEHealthcare). Scans were quantified with ImageQuant™ (GE Healthcare)software. Alternatively, in the absence of the inclusion of Cy5-labeleddCTP, the PCR products were separated on a 1.5% agarose gel containingtrace amounts of ethidium bromide for visualization.

PCR. PCR was performed with Platinum® Taq DNA Polymerase (Invitrogen)according to the manufacturer's directions. For each 50 μL reaction,approximately 30 pmol of both forward and reverse primers were used.Primers used in the examples described herein are included in Table 2.The thermocycling reaction proceeded, unless otherwise stated, asfollows: 94° C., 3 minutes; then 30-40 cycles of 94° C., 30 sec; 55° C.,30 sec; and 72° C.; 105 sec; followed by 72° C., 3 minutes. The PCRproducts were analyzed on 1.5% agarose gels and visualized with ethidiumbromide.

TABLE 2 RT-PCR and PCR Primers SEQ ID. Name Sequence 5′ to 3′Human TNFR2  74 TR001 ACT GGG CTT CAT CCC AGC ATC  75 TR002CAC CAT GGC GCC CGT CGC CGT  CTG G  76 TR003CGA CTT CGC TCT TCC AGT TGA  GAA GCC CTT GTG CCT GCA G  77 TR004TTA ACT GGG CTT CAT CCC AGC  ATC  78 TR005 CTG CAG GCA CAA GGG CTT CTC AAC TGG AAG AGC GAA GTC G  79 TR026 TTA ACT GGG CTT CAT CCC AGC  80TR027 CGA TAG AAT TCA TGG CGC CCG  TCG CCG TCT GG  81 TR028CCT AAC TCG AGT TAA CTG GGC  TTC ATC CCA GC  82 TR029GAC TGA GCG GCC GCC ACC ATG  GCG CCC GTC GCC GTC TGG  83 TR030CTA AGC GCG GCC GCT TAA CTG  GGC TTC ATC CCA GCA TC  84 TR047CGT TCT CCA ACA CGA CTT CA  85 TR048 CTT ATC GGC AGG CAA GTG AGG  86TR049 ACT GAA ACA TCA GAC GTG GTG  TGC  87 TR050CCT TAT CGG CAG GCA AGT GAG Human TNFR1  88 TR006CCT CAT CTG AGA AGA CTG GGC  G  89 TR007 GCC ACC ATG GGC CTC TCC ACC GTG C  90 TR008 GGG CAC TGA GGA CTC AGT TTG  TGG GAA ATC GAC ACC TG  91TR009 CAG GTG TCG ATT TCC CAC AAA  CTG AGT CCT CAG TGC CC  92 TR010CAC CAT GGG CCT CTC CAC CGT  GC  93 TR011 TCT GAG AAG ACT GGG CG  94TR031 CGA TAG GAT CCA TGG GCC TCT  CCA CCG TGC  95 TR032CCT AAC TCG AGT CAT CTG AGA  AGA CTG GGC G  96 TR033GAC TGA GCG GCC GCC ACC ATG  GGC CTC TCC ACC GTG C  97 TR034CTA AGC GCG GCC GCT CAT CTG  AGA AGA CTG GGC G Mouse TNFR2  98 TR012GGT CAG GCC ACT TTG ACT GC  99 TR013 CAC CGC TGC CCC TAT GGC G 100 TR014CAC CGC TGC CAC TAT GGC G 101 TR015 GGT CAG GCC ACT TTG ACT GCA  ATC 102TR016 GCC ACC ATG GCG CCC GCC GCC  CTC TGG 103 TR017GGC ATC TCT CTT CCA ATT GAG  AAG CCC TCC TGC CTA CAA AG 104 TR018CTT TGT AGG CAG GAG GGC TTC  TCA ATT GGA AGA GAG ATG CC 105 TR019GGC CAC TTT GAC TGC AAT CTG 106 TR035 CAC CAT GGC GCC CGC CGC CCT  CTG G107 TR036 TCA GGC CAC TTT GAC TGC AAT  C 108 TR037CGA TAG AAT TCA TGG CGC CCG  CCG CCC TCT GG 109 TR038CCT AAC TCG AGT CAG GCC ACT  TTG ACT GCA ATC 110 TR039GAC TGA GCG GCC GCC ACC ATG  GCG CCC GCC GCC CTC TGG 111 TR040CTA AGC GCG GCC GCT CAG GCC  ACT TTG ACT GCA ATC 112 TR045GAG CCC CAA ATG GAA ATG TGC 113 TR046 GCT CAA GGC CTA CTG CAT CCMouse TNFR1 114 TR020 GGT TAT CGC GGG AGG CGG GTC  G 115 TR021GCC ACC ATG GGT CTC CCC ACC  GTG CC 116 TR022CAC AAA CCC CCA GGA CTC AGT  TTG TAG GGA TCC CGT GCC T 117 TR023AGG CAC GGG ATC CCT ACA AAC  TGA GTC CTG GGG GTT TGT G 118 TR024CAC CAT GGG TCT CCC CAC CGT  GCC 119 TR025 TCG CGG GAG GCG GGT CGT GG120 TR041 CGA TAG TCG ACA TGG GTC TCC  CCA CCG TGC C 121 TR042CCT AAG AAT TCT TAT CGC GGG  AGG CGG GTC G 122 TR043GAC TGA GCG GCC GCC ACC ATG  GGT CTC CCC ACC GTG CC 123 TR044CTA AGC GCG GCC GCT TAT CGC  GGG AGG CGG GTC G

Human hepatocyte cultures. Human hepatocytes were obtained in suspensioneither from ADMET technologies, or from The UNC Cellular Metabolism andTransport Core at UNC-Chapel Hill. Cells were washed and suspended inRPMI 1640 supplemented with 10% FBS, 1 μg/mL human insulin, and 13 nMDexamethasone. Hepatocytes were plated in 6-well plates at 0.5×10⁶ cellsper plate in 3 mL media. After 1-1.5 hrs, non-adherent cells wereremoved, and the media was replaced with RPMI 1640 without PBS,supplemented with 1 μg/mL human insulin, and 130 nM Dexamethasone.

For delivery of SSOs to hepatocytes in 6-well plates, 10 μL of a 5 μMSSO stock was diluted into 100 μL of OPTI-MEM™, and 4 μL ofLipofectamine™ 2000 was diluted into 100 μL of OPTI-MEM™. The 200 μLcomplex solution was then applied to the cells in the 6-well platecontaining 2800 μL of media, for a total of 3000 μL. The final SSOconcentration was 17 nM. After 24 hrs, cells were harvested inTRI-Reagent™. Total RNA was isolated per the manufacturer's directions.Approximately 200 ng of total RNA was subjected to reversetranscription-PCR (RT-PCR).

ELISA. To determine the levels of soluble TNFR2 in cell culture media orsera, the Quantikine® Mouse sTNF RII ELISA kit from R&D Systems(Minneapolis, Minn.) or Quantikine® Human sTNF RII ELISA kit from R&DSystems (Minneapolis, Minn.) were used. The antibodies used fordetection also detect the protease cleavage forms of the receptor. ELISAplates were read using a microplate reader set at 450 nm, withwavelength correction set at 570 nm.

For mouse in vivo studies, blood from the animals was clotted for 1 hourat 37° C. and centrifuged for 10 min at 14,000 rpm (Jouan BRA4icentrifuge) at 4° C. Sera was collected and assayed according to themanufacturer's guide, using 50 μL of mouse sera diluted 1:10.

L929 cytotoxicity assay. L929 cells plated in 96-well plates at 10⁴cells per well were treated with 0.1 ng/mL TNF-α and 1 μg/mL actinomycinD in the presence of 10% serum from mice treated with the indicatedoligonucleotide in 100 μL total of complete MEM media (containing 10%regular FBS) and allowed to grow for ˜24 hrs at 37° C. Control laneswere plated in 10% serum from untreated mice. Cell viability wasmeasured 24 hrs later by adding 20 μl, CellTiter 96® AQ_(ueous) OneSolution Reagent (Promega) and measuring absorbance at 490 nm with amicroplate reader. Cell viability was normalized to untreated cells.

Western blots. Twenty μL of media or 20 μg of lysate were loaded in eachwell of a 4-12% NuPAGE® polyacrylamide gel (Invitrogen). The gel was run40 min at 200V. The protein was transferred, for 1 hr at 30V, to anInvitrolon™ PVDF membrane (Invitrogen), which was then blocked withStartingBlock® Blocking Buffer (Pierce) for 1 hr at room temperature.The membrane was incubated for 3 hrs at room temperature with a rabbitpolyclonal antibody that recognizes the C-terminus of human and mouseTNFR2 (Abeam), Following three washes in PBS-T buffer (1×PBS, 0.1%Tween-20), the membrane was incubated for one hour at room temperaturewith secondary goat anti-rabbit antibody (Abeam) and again washed threetimes with PBS-T buffer. The protein was then detected with ECL Plus™(GE Healthcare), according to the manufacturer's recommendations andthen photographed.

Example 2 SSO Splice Switching Activity with TNFR mRNA

Table 3 shows the splice switching activities of SSOs having sequencesas described in U.S. application Ser. No. 11/595,485 and targeted tomouse and human TNFRs. Of SSOs targeted to mouse TNFR2 exon 7, at least8 generated some muTNFR2 Δ7 mRNA. In particular, SSO 3312, 3274 and 3305induced at least 50% skipping of exon 7; SSO 3305 treatment resulted inalmost complete skipping. Of SSOs transfected into primary humanhepatocytes, and targeted to human TNFR2 exon 7, at least 7 SSOsgenerated some huTNFR2 Δ7 mRNA. In particular, SSOs 3378, 3379, 3384 and3459 induced at least 75% skipping of exon 7 (FIG. 2B), and significantinduction of huTNFR2 Δ7 into the extracellular media (FIG. 2A).

TABLE 3 SSO Splice Switching Activity SEQ ID. Name Activity Mouse TNFR23272 − 3304 − 3305 + 3306 + 3307 + 3308 + 3309 + 3310 − 3311 + 62 3274 +3312 + 3273 − Mouse TNFR1 3333 + Human TNFR2 14 3378 + 30 3379 + 3380 −70 3381 + 71 3382 + 3383 − 46 3384 + 72 3459 + 3460 − 73 3461 + Control3083 −

Table 4 contains the sequences of 10 nucleotide chimeric SSOs withalternating 2′O-methyl-ribonucleoside-phosphorothioates (2′-OMe) and2′O-4′-(methylene)-bicyclic-ribonucleoside phosphorothioates. These SSOsare targeted to exon 7 of mouse TNFR2.

TABLE 4 LNA/2′-OMe-ribonucleosidephosphorothioatechimeric mouse targeted SSO SEQ ID. Name Sequence 5′ to 3′* 62 3274AgAgCaGaAcCtTaCt 63 3837       gAaCcTuAcT 64 3838 aGaGcAgAaC 65 3839 gAgCaGaAcC 66 3840   aGcAgAaCcT 67 3841    gCaGaAcCuT 68 3842    cAgAaCcTuA 69 3843      aGaAcCuTaC *Capital letters are2′O-4′-(methylene)-bicyclic-ribonucleosides; lowercase letters are2′-OMe

To analyze the in vitro splice-switching activity of the SSOs listed inTable 4, L929 cells were cultured and seeded as described in Example 1.For delivery of each of the SSOs in Table 4 to the L929 cells, SSOs werediluted into 504 of OPTI-MEM™, and then 50 μL Lipofectamine™ 2000 mix (1part Lipofectamine™ 2000 to 25 parts OPTI-MEM™) was added and incubatedfor 20 minutes. Then 400 μL of serum free media was added to the SSOsand applied to the cells in the 24-well plates. The final SSOconcentration was either 50 or 100 nM. After 24 hrs, cells wereharvested in 800 μL TRI-Reagent™. Total RNA was isolated per themanufacturer's directions and analyzed by RT-PCR (FIG. 3) using theforward primer TR045 (SEQ ID No: 112) and the reverse primer TR046 (SEQID No: 113).

To analyze the in vivo splice-switching activity of the SSOs listed inTable 4, mice were injected with the SSOs listed in Table 4intraperitoneal (i.p.) at 25 mg/kg/day for 5 days. Mice were bled beforeinjection and again 1, 5 and 10 days after the Last injection. Theconcentration of soluble TNFR2 Δ7 in the sera taken before the firstinjection and 10 days after the last injection were measured by ELISA(FIG. 4B). The mice were sacrificed on day 10 and total RNA from 5-10 mgof the liver was analyzed by RT-PCR (FIG. 4A) using the forward primerTR045 (SEQ ID No: 112) and the reverse primer TR046 (SEQ ID No: 113).

Of the 10 nucleotide SSOs subsequences of SSO 3274 tested in vitro, allof them generated at least some muTNFR2 Δ7 mRNA (FIG. 3). In particular,SSO 3839, 3840 and 3841 displayed greater splice-switching activity thanthe longer 16 nucleotide SSO 3274 from which they are derived. The three10 nucleotide SSOs, 3839, 3840, 3841, that demonstrated the greatestactivity in vitro also were able to generate significant amounts ofmuTNFR2 Δ7 mRNA (FIG. 4A) and soluble muTNFR2 Δ7 protein (FIG. 4B) inmice in vivo.

To assess the effect of SSO length on splice switching activity in humanTNFR2, cells were treated with SSOs of different lengths. Primary humanhepatocytes were transfected with the indicated SSOs selected fromTable 1. These SSOs were synthesized by Santaris Pharma, Denmark withalternating 2′deoxy- and 2′O-4′-(methylene)-bicyclic-ribonucleosidephosphorothioates. For each SSO, the 5′-terminal nucleoside was a2′O-4′-methylene-ribonucleoside and the 3′-terminal nucleoside was a2′deoxy-ribonucleoside. These SSOs were either 10-, 12-, 14- or 16-mers.The concentration of soluble TNFR2 Δ7 was measured by ELISA (FIG. 5, toppanel). Total RNA was analyzed by RT-PCR for splice switching activity(FIG. 5, bottom panel).

Example 3 Analysis of the Splice Junction of SSO-Induced TNFR2 SpliceVariants

To confirm that the SSO splice switching, both in mice and in humancells, leads to the expected TNFR2 Δ7 mRNA, SSO-induced TNFR2 Δ7 mRNAwas analyzed by RT-PCR and was sequenced.

Mice. Mice were injected with SSO 3274 intraperitoneal (i.p.) at 25mg/kg/day for 10 days. The mice were then sacrificed and total RNA fromthe liver was analyzed by RT-PCR using the forward primer TR045 (SEQ IDNo: 112) and the reverse primer TR046 (SEQ ID No: 113). The productswere analyzed on a 1.5% agarose gel (FIG. 6A) and the product for theTNFR2 Δ7 was isolated using standard molecular biology techniques. Theisolated TNFR2 Δ7 product was amplified by PCR using the same primersand then sequenced (FIG. 6B). The sequence data contained the sequenceCTCTCTTCCAATTGAGAAGCCCTCCTGC (nucleotides 777-804 of SEQ ID No: 11),which confirms that the SSO-induced TNFR2 Δ7 mRNA lacks exon 7 and thatexon 6 is joined directly to exon 8.

Human hepatocytes. Primary human hepatocytes were transfected with SSO3379 as described in Example 1. Total RNA was isolated 48 hrs aftertransfection. The RNA was converted to cDNA with the Superscript™ HReverse Transcriptase (Invitrogen) using random hexamer primersaccording to the manufacturer's directions. PCR was performed on thecDNA using the forward primer TR049 (SEQ ID No: 86) and the reverseprimer TR050 (SEQ ID No: 87). The products were analyzed on a 1.5%agarose gel (FIG. 7A). The band corresponding to TNFR2 Δ7 was isolatedusing standard molecular biology techniques and then sequenced (FIG.7B). The sequence data contained the sequenceCGCTCTTCCAGTTGAGAAGCCCTTGTGC (nucleotides 774-801 of SEQ ID No: 9),which confirms that the SSO-induced TNFR2 Δ.7 mRNA lacks exon 7 and thatexon 6 is joined directly to exon 8.

Example 4 SSO Dose-Dependent Production of TNFR2 Δ7 Protein in PrimaryHuman Hepatocytes

The dose response of splice-switching activity of SSOs in primary humanhepatocytes was tested. Human hepatocytes were obtained in suspensionfrom ADMET technologies. Cells were washed three times and suspended inseeding media (RPMI 1640 supplemented with L-Glut, with 10% FBS,penicillin, streptomycin, and 12 nM Dexamethasone). Hepatocytes wereevaluated for viability and plated in 24-well, collagen-coated plates at1.0×10⁵ cells per well. Typically, cell viability was 85-93%. Afterapproximately 24 hrs, the media was replaced with maintenance media(seeding media without FES).

For delivery of each of the SSOs to the hepatocytes, SSOs were dilutedinto 50 μL of OPTI-MEM™, and then 50 μL Lipofectamine™ 2000 mix (1 partLipofectamine™ 2000 to 25 parts OPTI-MEM™) was added and incubated for20 minutes. The SSOs were then applied to the cells in the 24-wellplates. The final SSO concentration ranged from 1 to 150 nM. After 48hrs, cells were harvested in 800 μL TRI-Reagent™.

Total RNA from the cells was analyzed by RT-PCR using the forward primerTR047 (SEQ ID No: 84) and the reverse primer TR048 (SEQ ID No: 85) (FIG.8A). The concentration of soluble TNFR2 Δ7 in the serum was measured byELISA (FIG. 8B). Both huTNFR2 Δ7 mRNA (FIG. 8A) and secreted huTNFR2 Δ7protein (FIG. 8B) displayed dose dependent increases.

Example 5 Secretion of TNFR2 Splice Variants from Murine Cells

The ability of SSOs to induce soluble TNFR2 protein production andsecretion into the extracellular media was tested. L929 cells weretreated with SSOs as described in Example 1, and extracellular mediasamples were collected ˜48 hrs after transfection. The concentration ofsoluble TNFR2 in the samples was measured by ELISA (FIG. 9). SSOs thatbest induced shifts in RNA splicing, also secreted the most protein intothe extracellular media. In particular, SSOs 3305, 3312, and 3274increased soluble TNFR2 at least 3.5-fold over background. Consequently,induction of the splice variant mRNA correlated with production andsecretion of the soluble TNFR2.

Example 6 In Vivo Injection of SSOs Generated muTNFR2 Δ7 mRNA in Mice

SSO 3305 in saline was injected intraperitoneal (i.p.) daily for 4 daysinto mice at doses from 3 mg/kg to 25 mg/kg. The mice were sacrificed onday 5 and total RNA from the liver was analyzed by RT-PCR. The data showsplice switching efficacy similar to that found in cell culture. At themaximum dose of 25 mg/kg, SSO 3305 treatment induced almost fullconversion to Δ7 mRNA (FIG. 10, bottom panel).

A similar experiment with SSO 3274 induced about 20% conversion to Δ7mRNA. To optimize SSO 3274 induction of Δ7 mRNA, both the dose regimenand the time from the last injection to the sacrifice of the animal werevaried. SSO 3274 was injected (i.p.) into mice daily for 4 days. SSOtreatment induced about 30% conversion to Δ7 mRNA in mice analyzed onday 15, whereas a 20% shift was observed in mice analyzed on day five(FIG. 10, top panel). Furthermore, mice given injections for 10 days,and sacrificed on day 11 showed a 50% induction of Δ7 mRNA (FIG. 10,top). These in viva data suggest that TNFR2 SSOs can produce muTNFR2 Δ7mRNA for at least 10 days after administration.

Example 7 Circulatory TNFR2 Δ7

Mice were injected with SSO 3274, 3305, or the control 3083intraperitoneal (i.p.) at 25 mg/kg/day for 10 days. Mice were bledbefore injection and again 1, 5 and 10 days after the last injection.The concentration of soluble TNFR2 Δ7 in the serum was measured. SSOtreatment induced soluble TNFR2 Δ7 protein levels over background for atleast 10 days (FIG. 11).

To test the effects at longer time points, the experiment was repeated,except that serum samples were collected until day 27 after the lastinjection. The results show only a slight decrease in soluble TNFR2 Δ7levels 27 days after the last SSO injection (MG. 12).

Example 8

Anti-TNF-α Activity in Mice Serum

The anti-TNF-α activity of serum from SSO 3274 treated mice was testedin an L929 cytotoxicity assay. In this assay, serum is assessed for itsability to protect cultured L929 cells from the cytotoxic effects of afixed concentration of TNF-α as described in Example 1. Serum from micetreated with SSO 3274 but not control SSOs (3083 or 3272) increasedviability of the L929 cells exposed to 0.1 ng/mL TNF-α (FIG. 13). Hence,the SSO 3274 serum contained TNF-α antagonist sufficient to bind and toinactivate TNF-α, and thereby protect the cells from the cytotoxiceffects of INF-α. This anti-TNF-α activity was present in the serum ofanimals 5 and 27 days after the last injection of SSO 3274.

Example 9 Comparison of SSO Generated TNFR2 Δ7 to Other Anti-TNF-αAntagonists

L929 cells were seeded as in Example 8. Samples were prepared containing90 of serum-free MEM, 0.1 ng/ml TNF-α and 1 μg/ml of actinomycin D, witheither (i) recombinant soluble protein (0.01-3 μg/mL)) from Sigma®having the 236 amino acid residue extracellular domain of mouse TNFR2,(ii) serum from SSO 3274 or SSO 3305 treated mice (1.25-10%, diluted inserum from untreated mice; the concentration of TNFR2 Δ7 was determinedby ELISA) or (iii) Enbrel® (0.45-1.50 pg/ml) to a final volume of 100 μlwith a final mouse serum concentration of 10%. The samples wereincubated at room temperature for 30 minutes. Subsequently, the sampleswere applied to the plated cells and incubated for ˜24 hrs at 37° C. ina 5% CO₂ humidified atmosphere. Cell viability was measured by adding 20μL CellTiter 96® AQ_(ueous) One Solution Reagent (Promega) and measuringabsorbance at 490 nm with a microplate reader. Cell viability wasnormalized to untreated cells and plotted as a function of TNFantagonist concentration (FIG. 14).

Example 10 Stability of TNFR2 Δ7 mRNA and Protein

Mice were treated with either SSO 3274 or 3272 (control) (n=5) by i.p.injection at a dose of 25 mg/kg/day daily for five days. Mice were bledbefore injection and again 5, 15, 22, 27, and 35 days after the lastinjection. The concentration of soluble TNFR2 Δ7 in the serum wasmeasured (FIG. 15A). Splice shifting of TNFR2 in the liver was alsodetermined at the time of sacrifice by RT-PCR of total RNA from theliver (FIG. 15B). Combined with data from Example 7, a time course ofTNFR2 mRNA levels after SSO treatment was constructed, and compared withthe time course of TNFR2 Δ7 protein in serum (FIG. 16). The data showthat TNFR2 Δ7 mRNA in vivo decays at a rate approximately 4 times fasterthan that of TNFR2 Δ7 protein in serum. On day 35, TNFR2 Δ7 mRNA wasonly detectable in trace amounts, whereas TNFR2 Δ7 protein had onlydecreased by 20% from its peak concentration.

Example 11 Generation of Human TNFR2 Δ7 cDNA

A plasmid containing the full length human TNFR2 cDNA was obtainedcommercially from OriGene (Cat. No: TC119459, NM_001066.2). The cDNA wasobtained by performing PCR on the plasmid using reverse primer TR001(SEQ ID No: 74) and forward primer TR002 (SEQ ID No: 75). The PCRproduct was isolated and was purified using standard molecular biologytechniques, and contains the 1383 bp TNFR2 open reading frame without astop codon.

Alternatively, full length human TNFR2 cDNA is obtained by performingRT-PCR on total RNA from human mononuclear cells using the TR001 reverseprimer and the TR002 forward primer. The PCR product is isolated and ispurified using standard molecular biology techniques.

To generate human TNFR2 Δ7 cDNA, two separate PCR reactions wereperformed on the full length human TNFR2 cDNA, thereby creatingoverlapping segments of the TNFR2 Δ7 cDNA. In one reaction, PCR wasperformed on full length TNFR2 cDNA using the forward primer TR003 (SEQID No: 76) and the reverse primer TR004 (SEQ ID No: 77). In the otherreaction, PCR was performed on full length TNFR2 cDNA using the reverseprimer TR005 (SEQ ID No: 78) and the TR002 forward primer. Finally, the2 overlapping segments were combined, and PCR was performed using theTR002 forward primer and the TR004 reverse primer. The PCR product wasisolated and was purified using standard molecular biology techniques,and was expected to contain the 1308 bp TNFR2 Δ7 open reading frame witha stop codon (SEQ ID No: 9).

Similarly, by using the TR001 reverse primer instead of the TR004reverse primer in these PCR reactions the 1305 bp human TNFR2 Δ7 openreading frame without a stop codon was generated. This allows for theaddition of in-frame C-terminal affinity purification tags, such asHis-tag, when the final PCR product is inserted into an appropriatevector.

Example 12 Generation of Human TNFR1 Δ7 cDNA

A plasmid containing the full length human TNFR2 cDNA is obtainedcommercially from OriGene (Cat. No: TC127913, NM_001065.2). The cDNA isobtained by performing PCR on the plasmid using the TR006 reverse primer(SEQ ID No: 88) and the TR007 forward primer (SEQ ID No: 89). The fulllength human TNFR1 cDNA PCR product is isolated and is purified usingstandard molecular biology techniques.

Alternatively, full length human TNFR1 cDNA is obtained by performingRT-PCR on total RNA from human mononuclear cells using the TR006 reverseprimer and the TR007 forward primer. The full length human TNFR1 cDNAPCR product is isolated and is purified using standard molecular biologytechniques.

To generate human TNFR1 Δ7 cDNA, two separate PCR reactions areperformed on the full length human TNFR1 cDNA, thereby creatingoverlapping segments of the TNFR1 Δ7 cDNA. In one reaction, PCR isperformed on full length TNFR1 cDNA using the TR008 forward primer (SEQID No: 90) and the TR006 reverse primer. In the other reaction, PCR isperformed on full length TNFR1 cDNA using the TR009 reverse primer (SEQID No: 91) and the TR010 forward primer (SEQ ID No: 92). Finally, the 2overlapping segments are combined, and PCR is performed using the TR010forward primer and the TR006 reverse primer. The PCR product is isolatedand is purified using standard molecular biology techniques, andcontains the 1254 bp human TNFR1 Δ7 open reading frame with a stop codon(SEQ ID No: 5).

Alternatively, by using the TRW 1 reverse primer (SEQ ID No: 93) insteadof the TR006 reverse primer in these PCR reactions the 1251 bp humanTNFR1 Δ7 open reading frame without a stop codon is generated. Thisallows for the addition of in-frame C-terminal affinity purificationtags, such as His-tag, when the final PCR product is inserted into anappropriate vector.

Example 13 Generation of Murine TNFR2 Δ7 cDNA

To generate full length murine TNFR2 cDNA, PCR was performed on thecommercially available FirstChoice™ PCR-Ready Mouse Liver eDNA (Ambion,Cat. No: AM3300) using the TR012 reverse primer (SEQ ID No: 98) and theTR013 forward primer (SEQ ID No: 99). The full length murine TNFR2 cDNAPCR product is isolated and is purified using standard molecular biologytechniques. Then by performing PCR on the resulting product using theTR014 forward primer (SEQ ID No: 100) and the TR012 reverse primer theproper Kozak sequence was introduced.

Alternatively, full length murine TNFR2 cDNA is obtained by performingRT-PCR on total RNA from Mouse mononuclear cells or mouse hepatocytesusing the TR015 reverse primer (SEQ ID No: 101) and the TR016 forwardprimer (SEQ ID No: 102). The full length murine TNFR2 cDNA PCR productis isolated and is purified using standard molecular biology techniques.

To generate murine TNFR2 Δ7 cDNA, two separate PCR reactions wereperformed on the full length murine TNFR2 cDNA, thereby creatingoverlapping segments of the TNFR2 Δ7 cDNA. In one reaction, PCR wasperformed on full length TNFR2 cDNA using the TR017 forward primer (SEQID No: 103) and the TR015 reverse primer. In the other reaction, PCR wasperformed on full length TNFR2 cDNA using the TR018 reverse primer (SEQID No: 104) and the TR016 forward primer. Finally, the 2 overlappingsegments were combined, and PCR was performed using the TR016 forwardprimer and the TR015 reverse primer. The PCR product was isolated andwas purified using standard molecular biology techniques, and wasexpected to contain the 1348 bp murine TNFR2 Δ7-open reading frame witha stop codon (SEQ ID No: 11).

Alternatively, by using the TR019 reverse primer (SEQ ID No: 105)instead of the TR015 reverse primer in these PCR reactions the 1345 bpmurine TNFR2 Δ7 open reading frame without a stop codon was generated.This allows for the addition of in-frame C-terminal affinitypurification tags, such as His-tag, when the final PCR product isinserted into an appropriate vector.

Example 14 Generation of Murine TNFR1 Δ7 cDNA

To generate full length murine TNFR1 cDNA, PCR is performed on thecommercially available FirstChoice™ PCR-Ready Mouse Liver cDNA (Ambion,Cat. No: AM3300) using the TR020 reverse primer (SEQ ID No: 114) and theTR021 forward primer (SEQ ID No: 115). The full length murine TNFR1 cDNAPCR product is isolated and is purified using standard molecular biologytechniques.

Alternatively, full length murine TNFR1 cDNA is obtained by performingRT-PCR on total RNA from mouse mononuclear cells using the TR020 reverseprimer and the TR021 forward primer. The full length murine TNFR1 cDNAPCR product is isolated and is purified using standard molecular biologytechniques.

To generate murine TNFR1 Δ7 cDNA, two separate PCR reactions areperformed on the full length human TNFR1 cDNA, thereby creatingoverlapping segments of the TNFR1 Δ7 cDNA. In one reaction, PCR isperformed on full length TNFR1 cDNA using the TR022 forward primer (SEQID No: 116) and the TR020 reverse primer. In the other reaction, PCR isperformed on full length TNFR1 cDNA using the TR023 reverse primer (SEQID No: 117) and the TR024 forward primer (SEQ ID No: 118). Finally, the2 overlapping segments are combined, and PCR is performed using TR024forward primer and the TR020 reverse primer. The 1259 bp PCR product isisolated and is purified using standard molecular biology techniques,and contains the 1251 bp murine TNFR1 Δ7 open reading frame with a stopcodon (SEQ ID No: 7).

Alternatively, by using the TR025 reverse primer (SEQ ID No: 119)instead of the TR020 reverse primer in these PCR reactions the 1248 bpmurine TNFR1 Δ7 open reading frame without a stop codon is generated.This allows for the addition of in-frame C-terminal affinitypurification tags, such as His-tag, when the final PCR product isinserted into an appropriate vector.

Example 15 Construction of Vectors for the Expression of Human TNFR2 Δ7in Mammalian Cells

For expression of the human TNFR2 Δ7 protein in mammalian cells, a humanTNFR2 Δ7 cDNA PCR product from Example 11 was incorporated into anappropriate mammalian expression vector. The TNFR2 Δ7 cDNA PCR productfrom Example 11, both with and without a stop codon, and the pcDNA™3.1D/V5-His TOPO® expression vector (Invitrogen) were blunt-end ligatedand isolated according to the manufacturer's directions. Plasmidscontaining inserts encoding human TNFR2 Δ7 were transformed intoOneShot® Top10 competent cells (Invitrogen), according to the supplier'sdirections. Fifty μL of the transformation mix were plated on LB mediawith 100 μg/mL of ampicillin and incubated overnight at 37° C. Singlecolonies were used to inoculate 5 mL cultures of LB media with 100 μg/mLampicillin and incubated overnight at 37° C. The cultures were then usedto inoculate 200 mL of LB media with 100 μg/mL of ampicillin and grownovernight at 37° C. The plasmids were isolated using GenElute™ PlasmidMaxiprep kit (Sigma) according to manufacturer's directions.Purification efficiency ranged from 0.5 to 1.5 mg of plasmid perpreparation.

Three human TNFR2 Δ7 clones (1319-1, 1138-5 and 1230-1) were generatedand sequenced. Clone 1319-1 contains the human TNFR2 Δ7 open readingframe without a stop codon followed directly by an in-frame His-tag fromthe plasmid; while clones 1138-5 and 1230-1 contain the TNFR2 Δ7 openreading frame followed immediately by a stop codon. The sequence of theHis-tag from the plasmid is given in SEQ ID No: 126. The sequences ofthe TNFR2 Δ7 open reading frames of clones 1230-1 and 1319-1 wereidentical to SEQ ID No: 9 with and without the stop codon, respectively.However relative to SEQ ID No: 9, the sequence (SEQ ID No: 125) of theTNFR2 Δ7 open reading frames of clone 1138-5 differed by a singlenucleotide at position 1055 in exon 10, with an A in the former and a Gin the later. This single nucleotide change causes the amino acid 352 tochange from a glutamine to an arginine.

Example 16 Expression of Human TNFR2 Δ7 in E. coli

For expression of the human TNFR2 Δ7 protein in bacteria, a human TNFR2Δ7 cDNA from Example 11 is incorporated into an appropriate expressionvector, such as a pET Directional TOPO® expression vector (Invitrogen).PCR is performed on the PCR fragment from Example 11 using forward(TR002) (SEQ ID No: 75) and reverse (TR026) (SEQ ID No: 79) primers toincorporate a homologous recombination site for the vector. Theresulting PCR fragment is incubated with the pET101/D-TOPO® vector(Invitrogen) according to the manufacturer's directions, to create thehuman TNFR2 Δ7 bacterial expression vector. The resulting vector istransformed into the E. coli strain BL21(DE3). The human TNFR2 Δ7 isthen expressed from the bacterial cells according to the manufacturer'sinstructions.

Example 17 Expression of Human TNFR2 Δ7 in Insect Cells

For expression of the human TNFR2 Δ7 protein in insect cells, a humanTNFR2 Δ7 cDNA from Example 11 is incorporated into a baculoviral vector.PCR is performed on a human TNFR2 Δ7 cDNA from Example 11 using forward(TR027) (SEQ ID No: 80) and reverse (TR028) (SEQ ID No: 81) primers. Theresulting PCR product is digested with the restriction enzymes EcoRI andXhoI. The digested PCR product is ligated with a EcoRI and XhoI digestedpENTR™ Vector (Invitrogen), such as any one of the pENTR™1A, pENTR™2B,pENTR™3C, pENTR™4, or pENTR™11 Vectors, to yield an entry vector. Theproduct is then isolated, amplified, and purified using standardmolecular biology techniques.

A baculoviral vector containing the human TNFR2 Δ7 cDNA is generated byhomologous recombination of the entry vector with BaculoDirect™ LinearDNA (Invitrogen) using LR Clonase™ (Invitrogen) according to themanufacturer's directions. The reaction mixture is then used to infectSf9 cells to generate recombinant baculovirus. After harvesting therecombinant baculovirus, expression of human TNFR2 Δ7 is confirmed.Amplification of the recombinant baculovirus yields a high-titer viralstock. The high-titer viral stock is used to infect Sf9 cells, therebyexpressing human TNFR2 Δ7 protein.

Example 18 Generation of Adeno-Associated Viral Vectors for theExpression of Human TNFR2 Δ7

For in vitro or in vivo delivery to mammalian cells of the human TNFR2Δ7 gene for expression in those mammalian cells, a recombinantadeno-associated virus (rAAV) vector is generated using a three plasmidtransfection system as described in Grieger, J., et al., 2006, NatureProtocols 1:1412. PCR is performed on a purified human TNFR2 Δ7 PCRproduct of Example 11, using forward (TR029) (SEQ ID No: 82) and reverse(TR030) (SEQ ID No: 83) primers to introduce unique flanking NotIrestriction sites. The resulting PCR product is digested with the NotIrestriction enzyme, and isolated by standard molecular biologytechniques. The NotI-digested fragment is then ligated to NotI-digestedpTR-UF2 (University of North Carolina (UNC) Vector Core Facility), tocreate a plasmid that contains the human TNFR2 Δ7 open reading frame,operably linked to the CMVie promoter, flanked by inverted terminalrepeats. The resulting plasmid is then transfected with the plasmidspXX680 and pHelper (UNC Vector Core Facility) into HEK-293 cells, asdescribed in Grieger, J., et al., to produce rAAV particles containingthe human TNFR2 Δ7 gene where expression is driven by the strongconstitutive CMVie promoter. The virus particles are harvested andpurified, as described in Grieger, J., et al., to provide an rAAV stocksuitable for transducing mammalian cells.

Example 19 Expression of Human TNFR1 Δ7 in E. coli

For expression of the human TNFR1 Δ7 protein in bacteria, the cDNA fromExample 12 is incorporated into an appropriate expression vector, suchas a pET Directional TOPO® expression vector (Invitrogen). PCR isperformed on the cDNA from Example 12 using forward (TR010) (SEQ ID No:92) and reverse (TR006) (SEQ ID No: 88) primers to incorporate ahomologous recombination site for the vector. The resulting PCR fragmentis incubated with the pET101/D-TOPO® vector (Invitrogen) according tothe manufacturer's directions, to create the human TNFR1 Δ7 bacterialexpression vector. The resulting vector is transformed into the E. colistrain BL21(DE3). The human TNFR1 Δ7 is then expressed from thebacterial cells according to the manufacturer's instructions.

Example 20 Expression of Human TNFR1 Δ7 in Mammalian Cells

For expression of the human TNFR1 Δ7 protein in mammalian cells, a humanTNFR1 Δ7 cDNA PCR product from Example 12 is incorporated into anappropriate mammalian expression vector. human TNFR1 Δ7 cDNA PCR productfrom Example 12 and the pcDNA™3.1D/V5-His TOPO® expression vector(Invitrogen) are blunt-end ligated according to the manufacturer'sdirections. The product is then isolated, amplified, and purified usingstandard molecular biology techniques to yield the mammalian expressionvector. The vector is then transfected into a mammalian cell, whereexpression of the human TNFR1 Δ7 protein is driven by the strongconstitutive CMVie promoter.

Example 21 Expression of Human TNFR1 Δ7 in Insect Cells

For expression of the human TNFR1 Δ7 protein in insect cells, the cDNAfrom Example 12 is incorporated into a baculoviral vector. PCR isperformed on the cDNA from Example 12 using forward (TR031) (SEQ ID No:94) and reverse (TR032) (SEQ ID No: 95) primers. The resulting PCRproduct is digested with the restriction enzymes EcoRI and XhoI. Thedigested PCR product is ligated with a EcoRI and XhoI digested pENTR™Vector (Invitrogen), such as any one of the pENTR™1A, pENTR™2B,pENTR™3C, pENTR™4, or pENTR™11 Vectors, to yield an entry vector. Theproduct is then isolated, amplified, and purified using standardmolecular biology techniques.

A baculoviral vector containing the human TNFR1 Δ7 cDNA is generated byhomologous recombination of the entry vector with BaculoDirect™ LinearDNA (Invitrogen) using LR Clonaser™ (Invitrogen) according to themanufacturer's directions. The reaction mixture is then used to infectSf9 cells to generate recombinant baculovirus. After harvesting therecombinant baculovirus, expression of human TNFR1 Δ7 is confirmed.Amplification of the recombinant baculovirus yields a high-titer viralstock. The high-titer viral stock is used to infect Sf9 cells, therebyexpressing human TNFR1 Δ7 protein.

Example 22 Generation of Adeno-Associated Viral Vectors for theExpression of Human TNFR1 Δ7

For in vitro or in vivo delivery to mammalian cells of the human TNFR1Δ7 gene for expression in those mammalian cells, a recombinantadeno-associated virus (rAAV) vector is generated using a three plasmidtransfection system as described in Grieger, J., et al., 2006, NatureProtocols 1:1412. PCR is performed on the purified human TNFR1 Δ7 PCRproduct of Example 12, using forward (TR033) (SEQ ID No: 96) and reverse(TR034) (SEQ ID No: 97) primers to introduce unique flanking NotIrestriction sites. The resulting PCR product is digested with the NotIrestriction enzyme, and isolated by standard molecular biologytechniques. The NotI-digested fragment is then ligated to NotI-digestedpTR-UF2 (University of North Carolina (UNC) Vector Core Facility), tocreate a plasmid that contains the human TNFR1 Δ7 open reading frame,operably linked to the CMVie promoter, flanked by inverted terminalrepeats. The resulting plasmid is then transfected with the plasmidspXX680 and pHelper (UNC Vector Core Facility) into HEK-293 cells, asdescribed in Grieger, J., et al., to produce rAAV particles containingthe human TNFR1 Δ7 gene where expression is driven by the strongconstitutive CMVie promoter. The virus particles are harvested andpurified, as described in Grieger, J., et al., to provide an rAAV stocksuitable for transducing mammalian cells.

Example 23 Construction of Vectors for the Expression of Mouse TNFR2 Δ7in Mammalian Cells

For expression of the murine TNFR2 Δ7 protein in mammalian cells, amurine TNFR2 Δ7 cDNA PCR product from Example 13 was incorporated intoan appropriate mammalian expression vector. The TNFR2 Δ7 cDNA PCRproduct from Example 13, both with and without a stop codon, and thepcDNA™3.1D/V5-His TOPO® expression vector (Invitrogen) was blunt-endligated and isolated according to the manufacturer's directions.Plasmids containing inserts encoding murine Δ7 TNFR2 were transformedinto OneShot® Top10 competent cells (Invitrogen), according to thesupplier's directions. Fifty μL of the transformation mix were plated onLB media with 100 μg/mL of ampicillin and incubated overnight at 37° C.Single colonies were used to inoculate 5 mL cultures of LB media with100 fig/mL ampicillin and incubated overnight at 37° C. The cultureswere then used to inoculate 200 mL of LB media with 100 μg/mL ofampicillin and grown overnight at 37° C. The plasmids were isolatedusing GenElute™ Plasmid Maxiprep kit (Sigma) according to manufacturer'sdirections. Purification efficiency ranged from 0.5 to 1.5 mg of plasmidper preparation.

Two murine TNFR2 Δ7 clones (1144-4 and 1145-3) were generated andsequenced. Clone 1144-4 contains the murine TNFR2 Δ7 open reading framewithout a stop codon followed directly by an in-frame His-tag from theplasmid; while clone 1145-3 contains the TNFR2 Δ7 open reading framefollowed immediately by a stop codon. The sequence of the His-tag fromthe plasmid is given in SEQ ID No: 126. Relative to SEQ ID No: 11, thesequence (SEQ ID No: 124) of the TNFR2 Δ7 open reading frames of the twoclones, 1144-4 and 1145-3, differed by a single nucleotide at elevenpositions. As a result of these single nucleotide changes there are fouramino acid differences relative to SEQ ID No: 12.

Example 24 Expression of Murine TNFR2 Δ7 in E. coli

For expression of the mouse TNFR2 Δ7 protein in bacteria, a murine TNFR2Δ7 cDNA from Example 13 is incorporated into an appropriate expressionvector, such as a pET Directional TOPO® expression vector (Invitrogen).PCR is performed on the PCR fragment from Example 13 using forward(TR035) (SEQ ID No: 106) and reverse (TR036) (SEQ ID No: 107) primers toincorporate a homologous recombination site for the vector. Theresulting PCR fragment is incubated with the pET101/D-TOPO® vector(Invitrogen) according to the manufacturer's directions, to create themarine TNFR2 Δ7 bacterial expression vector. The resulting vector istransformed into the E. coli strain BL21(DE3). The murine TNFR2 Δ7 isthen expressed from the bacterial cells according to the manufacturer'sinstructions.

Example 25 Expression of Mouse TNFR2 Δ7 in Insect Cells

For expression of the murine TNFR2 Δ7 protein in insect cells, the cDNAfrom Example 13 is incorporated into a baculoviral vector. PCR isperformed on the cDNA from Example 13 using forward (TR037) (SEQ ID No:108) and reverse (TR038) (SEQ ID No: 109) primers. The resulting PCRproduct is digested with the restriction enzymes EcoRI and XhoI. Thedigested PCR product is ligated with a EcoRI and XhoI digested pENTR™Vector (Invitrogen), such as any one of the pENTR™1A, pENTR™2B,pENTR™3C, pENTR™4, or pENTR™11 Vectors, to yield an entry vector. Theproduct is then isolated, amplified, and purified using standardmolecular biology techniques.

A baculoviral vector containing the marine TNFR2 Δ7 cDNA is generated byhomologous recombination of the entry vector with BaculoDirect™ LinearDNA (Invitrogen) using LR Clonase™ (Invitrogen) according to themanufacturer's directions. The reaction mixture is then used to infectSf9 cells to generate recombinant baculovirus. After harvesting therecombinant baculovirus, expression of murine TNFR2 Δ7 is confirmed.Amplification of the recombinant baculovirus yields a high-titer viralstock. The high-titer viral stock is used to infect Sf9 cells, therebyexpressing murine TNFR2 Δ7 protein.

Example 26 Generation of Adeno-Associated Viral Vectors for theExpression of Murine TNFR2 Δ7

For in vitro or in vivo delivery to mammalian cells of the murine TNFR2Δ7 gene for expression in those mammalian cells, a recombinantadeno-associated virus (rAAV) vector is generated using a three plasmidtransfection system as described in Grieger, J., et al., 2006, NatureProtocols 1:1412. PCR is performed on the purified murine TNFR2 Δ7 PCRproduct of Example 13, using forward (TR039)(SEQ ID No: 110) and reverse(TR040)(SEQ ID No: 111) primers to introduce unique flanking NotIrestriction sites. The resulting PCR product is digested with the NotIrestriction enzyme, and isolated by standard molecular biologytechniques. The NotI-digested fragment is then ligated to NotI-digestedpTR-UF2 (University of North Carolina (UNC) Vector Core Facility), tocreate a plasmid that contains the murine TNFR2 Δ7 open reading frame,operably linked to the CMVie promoter; flanked by inverted terminalrepeats. The resulting plasmid is then transfected with the plasmidspXX680 and pHelper (UNC Vector Core Facility) into HEK-293 cells, asdescribed in Grieger, J., et al., to produce rAAV particles containingthe murine TNFR2 Δ7 gene where expression is driven by the strongconstitutive CMVie promoter. The virus particles are harvested andpurified, as described in Grieger, J., et al., to provide an rAAV stocksuitable for transducing mammalian cells.

Example 27 Expression of Murine TNFR1 Δ7 in E. coli

For expression of the mouse TNFR1 Δ7 protein in bacteria, the cDNA fromExample 14 is incorporated into an appropriate expression vector, suchas a pET Directional TOPO® expression vector (Invitrogen). PCR isperformed on the cDNA from Example 14 using forward (TR024)(SEQ ID No:118) and reverse (TR020)(SEQ ID No: 114) primers to incorporate ahomologous recombination site for the vector. The resulting PCR fragmentis incubated with the pET101/D-TOPO® vector (Invitrogen) according tothe manufacturer's directions, to create the murine TNFR1 Δ7 bacterialexpression vector. The resulting vector is transformed into the E. colistrain BL21(DE3). The murine TNFR1 Δ7 is then expressed from thebacterial cells according to the manufacturer's instructions.

Example 28 Expression of Mouse TNFR1 Δ7 in Mammalian Cells

For expression of the murine TNFR1 Δ7 protein in mammalian cells, amurine TNFR1 Δ7 cDNA PCR product from Example 14 is incorporated into anappropriate mammalian expression vector. The murine TNFR1 Δ7 cDNA PCRproduct from Example 14 and the pcDNA™3.1D/V5-His TOPO® expressionvector (Invitrogen) are blunt-end ligated according to themanufacturer's directions. The product is then isolated, amplified, andpurified using standard molecular biology techniques to yield themammalian expression vector. The vector is then transfected into amammalian cell, where expression of the murine TNFR1 Δ7 protein isdriven by the strong constitutive CMVie promoter.

Example 29 Expression of Mouse TNFR1 Δ7 in Insect Cells

For expression of the murine TNFR1 Δ7 protein in insect cells, the cDNAfrom Example 14 is incorporated into a baculoviral vector. PCR isperformed on the cDNA from Example 14 using forward (TR041)(SEQ ID No:120) and reverse (TR042) (SEQ ID No: 121) primers. The resulting PCRproduct is digested with the restriction enzymes EcoRI and XhoI. Thedigested PCR product is ligated with a EcoRI and XhoI digested pENTR™Vector (Invitrogen), such as any one of the pENTR™1A, pENTR™2B,pENTR™3C, pENTR™4, or pENTR™11 Vectors, to yield an entry vector. Theproduct is then isolated, amplified, and purified using standardmolecular biology techniques.

A baculoviral vector containing the murine TNFR1 Δ7 cDNA is generated byhomologous recombination of the entry vector with BaculoDirect™ LinearDNA (Invitrogen) using LR Clonase™ (Invitrogen) according to themanufacturer's directions. The reaction mixture is then used to infectSf9 cells to generate recombinant baculovirus. After harvesting therecombinant baculovirus, expression of murine TNFR1 Δ7 is confirmed.Amplification of the recombinant baculovirus yields a high-titer viralstock. The high-titer viral stock is used to infect Sf9 cells, therebyexpressing murine TNFR1 Δ7 protein.

Example 30 Generation of Adeno-Associated Viral Vectors for theExpression of Murine TNFR1 Δ7

For in vitro or in vivo delivery to mammalian cells of the murine TNFR1Δ7 gene for expression in those mammalian cells, a recombinantadeno-associated virus (rAAV) vector is generated using a three plasmidtransfection system as described in Grieger, J., et al., 2006, NatureProtocols 1:1412. PCR is performed on the purified murine TNFR1 Δ7 PCRproduct of Example 13, using forward (TR043)(SEQ ID No: 122) and reverse(TR044)(SEQ ID No: 123) primers to introduce unique flanking NotIrestriction sites. The resulting PCR product is digested with the NotIrestriction enzyme, and isolated by standard molecular biologytechniques. The NotI-digested fragment is then ligated to NotI-digestedpTR-UF2 (University of North Carolina (UNC) Vector Core Facility), tocreate a plasmid that contains the murine TNFR1 Δ7 open reading frame,operably linked to the CMVie promoter, flanked by inverted terminalrepeats. The resulting plasmid is then transfected with the plasmidspXX680 and pHelper (UNC Vector Core Facility) into HEK-293 cells, asdescribed in Grieger, J., et al., to produce rAAV particles containingthe murine TNFR1 Δ7 gene where expression is driven by the strongconstitutive CMVie promoter. The virus particles are harvested andpurified, as described in Grieger, J., et al., to provide an rAAV stocksuitable for transducing mammalian cells.

Example 31 Generation of Lentiviral Vectors for the Expression of TNFRΔ7

For in vitro or in vivo delivery to mammalian cells of a TNFR Δ7 genefor expression in those mammalian cells, a replication-incompetentlentivirus vector is generated. A PCR product from Example 16, Example19, Example 24 or Example 27 and the pLenti6/V5-D-TOPO® vector(Invitrogen) are blunt-end ligated according to the manufacturer'sdirections. The resulting plasmid is transformed into E. coli,amplified, and purified using standard molecular biology techniques.This plasmid is transfected into 293FT cells (Invitrogen) according tothe manufacturer's directions to produce lentivirus particles containingthe TNFR Δ7 gene where expression is driven by the strong constitutiveCMVie promoter. The virus particles are harvested and purified, asdescribed in Tiscornia, G., et al., 2006, Nature Protocols 1:241, toprovide a lentiviral stock suitable for transducing mammalian cells.

Example 32 Expression of TNFR2 Δ7 in Mammalian Cells

The plasmids generated in Example 15 and Example 23 were used to expressactive protein in mammalian HeLa cells, and the resulting proteins weretested for anti-TNF-α activity. HeLa cells were seeded in at 1.0×10⁵cells per well in 24-well plates in SMEM media containing L-glutamine,gentamicin, kanamycin, 5% FBS and 5% HS. Cells were grown overnight at37° C. in a 5% CO₂ humidified atmosphere. Approximately 250 ng ofplasmid DNA was added to 50 μL of OPTI-MEM™, and then 50 μLLipofectamine™ 2000 mix (1 part Lipofectamine™ 2000 to 25 partsOPTI-MEM™) was added and incubated for 20 minutes. Then 400 μL of serumfree media was added and then applied to the cells in the 24-wellplates. After incubation for ˜48 hrs at 37° C. in a 5% CO₂ humidifiedatmosphere, the media was collected and the cells were harvested in 800μL TRI-Reagent™. Total RNA was isolated from the cells per themanufacturer's directions and analyzed by RT-PCR using the forwardprimer TR047 (SEQ ID No: 84) and the reverse primer TR048 (SEQ ID No:85) for human TNFR2 Δ7, or the forward primer TR045 (SEQ ID No: 112) andthe reverse primer TR046 (SEQ ID No: 113) for mouse TNFR2 Δ7. Theconcentration of soluble TNFR2 in the media was measured by ELISA.

The anti-TNF-α activity of the above media was tested in an L929cytotoxicity assay. L929 cells were plated in 96-well plates at 2×10⁴cells per well in MEM media containing 10% regular FBS, penicillin andstreptomycin and grown overnight at 37° C. in a 5% CO₂ humidifiedatmosphere. The media samples were diluted 1, 2, 4, 8 and 16 fold withmedia from non-transfected HeLa cells. Ninety of each of these sampleswas added to 10 μL of serum-free media, containing 1.0 ng/ml TNF-α and 1μg/ml of actinomycin D. The media from the cells were removed andreplaced with these 100 μL samples. The cells were then grown overnightat 37° C. in a 5% CO₂ humidified atmosphere. Twenty μL CellTiter 96®AQ_(ueous) One Solution Reagent (Promega) was then added to each well.Cell viability was measured 4 hrs later by measuring absorbance at 490nm with a microplate reader. Cell viability was normalized to untreatedcells nd plotted as a function of TNF antagonist concentration (FIG.17).

The data from this example and from Example 9 were analyzed using theGraphPad Prism® software to determine the EC₅₀ value for eachantagonist. For each antagonist from these examples a sigmoidaldose-response curve was fit by non-linear regression with the maximumand minimum responses held fixed to 100% and 0%, respectively. The EC₅₀values shown in Table 5 correspond to a 95% confidence level, and eachcurve had an r² value ranging from 0.7 to 0.9.

TABLE 5 Activity of TNF-α antagonists TNF-α Antagonist EC₅₀ (ng/mL)Etanercept 1.1 ± 0.5 Recombinant soluble TNFR2 (rsTNFR2) 698 ± 180 SSO3305 treated mice serum (mouse TNFR2 Δ7) 0.6 ± 0.2 SSO 3274 treated miceserum (mouse TNFR2 Δ7) 0.8 ± 0.3 Extracellular media from 1144-4transfected HeLa 2.4 ± 1.4 cells (mouse TNFR2 Δ7) Extracellular mediafrom 1145-3 transfected HeLa 2.4 ± 0.8 cells (mouse TNFR2 Δ7)Extracellular media from 1230-1 transfected HeLa 1.4 ± 1.1 cells (humanTNFR2 Δ7) Extracellular media from 1319-1 transfected HeLa 1.7 ± 1.0cells (human TNFR2 Δ7) Extracellular media from 1138-5 transfected HeLa1.8 ± 1.1 cells (human TNFR2 Δ7)

Example 33 Expression and Purification of TNFR2 Δ7 in Mammalian Cells

The plasmids generated in Example 15 and Example 23 were used to expressand purify TNFR2 Δ7 from mammalian HeLa cells. HeLa cells were plated in6-well plates at 5×10⁵ cells per well, and grown overnight at 37° C., 5%CO₂, in humidified atmosphere. Each well was then transfected with 1.5μg of plasmid DNA using either 1144-4 (mouse TNFR2 Δ7 with His-tag),1145-1 (mouse TNFR2 Δ7, no His-tag), 1230-1 (human TNFR2 Δ7, no His-tag)or 1319-1 (human TNFR2 Δ7 with His-tag) plasmids. Media was collected˜48 hrs after transfection and concentrated approximately 40-fold usingAmicon MWCO 30,000 filters. The cells were lysed in 120 μL of RIM lysisbuffer (Invitrogen) with protease inhibitors (Sigma-aldrich) for 5minutes on ice. Protein concentration was determined by the Bradfordassay. Proteins were then isolated from aliquots of the cell lysates andthe extracellular media and analyzed by western blot for TNFR2 asdescribed in Example 1 (FIG. 18).

Human and mouse TNFR2 Δ7 with a His-tag (clones 1319-1 and 1144-4,respectively) were purified from the above media by affinitychromatography. HisPur™ cobalt spin columns (Pierce) were used to purifymouse and human TNFR2 Δ7 containing a His-tag from the above media.Approximately 32 mL of media were applied to a 1 mL HisPur™ columnequilibrated with 50 mM sodium phosphate, 300 mM sodium chloride, 10 mMimidazole buffer (pH 7.4) as recommended by the manufacturer. The columnwas then washed with two column volumes of the same buffer and proteinwas eluted with 1 mL of 50 mM sodium phosphate, 300 mM sodium chloride,150 mM imidazole buffer (pH 7.4). Five μL of each eluate were analyzedby Western blot as described above (FIG. 19). TNFR2 Δ7 appears in theeluate and the multiple bands represent variably glycosylated forms ofTNFR2 Δ7. As negative controls, the TNFR2 Δ7 proteins expressed fromplasmids 1230-1 or 1145-1 which do not contain a His-tag where subjectedto the above purification procedure. These proteins do not bind theaffinity column and do not appear in the eluate (FIG. 19).

1. An isolated protein capable of binding tumor necrosis factor (TNF),said protein having a sequence comprising the amino acids encoded by acDNA derived from a mammalian tumor necrosis factor receptor (TNFR)gene, wherein the cDNA comprises in 5′ to 3′ contiguous order, the codonencoding the first amino acid after the cleavage point of the signalsequence of said gene through exon 6 of said gene and exon 8 or saidgene through exon 10 of said gene; or the codon encoding the first aminoacid of the open reading frame of said gene through exon 6 of said geneand exon 8 of said gene through exon 10 of said gene. 2.-56. (canceled)